A promising pipeline of preclinical drug candidates for leishmaniasis and chronic Chagas' disease.

The drug discovery pipeline for leishmaniasis and trypanosomiasis has been filling with novel chemical entities with known mechanisms of action. González et al. and Braillard et al. report a cytochrome bc1 complex inhibitor as another promising preclinical candidate for visceral leishmaniasis (VL) and, in combination with benznidazole, for chronic Chagas' disease (CCD).

Parasite Burden of Trypanosoma cruzi in Whole Blood and Buffy Coat Determined by Real-Time PCR in Individuals with Chronic Chagas Disease.

The objective of this study was to compare, by qPCR, the circulating blood parasite load of Trypanosoma cruzi in the buffy coat, and in whole blood mixed with boiled and unboiled guanidine hydrochloride-EDTA buffer, of individuals with chronic ChD. The concentration and purity of DNA were evaluated in a Nanodrop Denovix DS-11FX Series Spectrophotometer (DeNovix Inc., Wilmington, NC, USA). The parasite load was determined with the Taqman® qPCR system using a Stratagene Mx3000P thermocycler (Agilent Technologies, Santa Clara, CA, USA) with Cruzi 1 and Cruzi 2 satellite primers. Student's t-test with Bonferroni correction, Chi-squared (χ2) tests and Spearman's correlation coefficient were applied. The concentration and purity of DNA were higher in the buffy coat. Parasite DNA was detected and quantifiable in the three types of samples in seven patients, without statistically significant differences in the parasite load obtained. Higher correlations were found between the total DNA concentrations and the parasite loads obtained in the samples of the buffy coat.

Exploring Peripheral Blood-Derived Extracellular Vesicles as Biomarkers: Implications for Chronic Chagas Disease with Viral Infection or Transplantation.

Extracellular vesicles (EVs) are lipid bilayer envelopes that encapsulate cell-specific cargo, rendering them promising biomarkers for diverse diseases. Chagas disease, caused by the parasite Trypanosoma cruzi, poses a significant global health burden, transcending its initial epicenter in Latin America to affect individuals in Europe, Asia, and North America. In this study, we aimed to characterize circulating EVs derived from patients with chronic Chagas disease (CCD) experiencing a reactivation of acute symptoms. Blood samples collected in EDTA were processed to isolate plasma and subsequently subjected to ultracentrifugation for particle isolation and purification. The EVs were characterized using a nanoparticle tracking analysis and enzyme-linked immunosorbent assay (ELISA). Our findings revealed distinctive differences in the size, concentration, and composition of EVs between immunosuppressed patients and those with CCD. Importantly, these EVs play a critical role in the pathophysiology of Chagas disease and demonstrate significant potential as biomarkers in the chronic phase of the disease. Overall, our findings support the potential utility of the CL-ELISA assay as a specific sensitive tool for detecting circulating EVs in chronic Chagasic patients, particularly those with recurrent infection following an immunosuppressive treatment or with concurrent HIV and Chagas disease. Further investigations are warranted to identify and validate the specific antigens or biomarkers responsible for the observed reactivity in these patient groups, which may have implications for diagnosis, the monitoring of treatment, and prognosis.

Seroepidemiology of Chagas disease in at-risk individuals in Caraíbas, a city with high endemicity in Bahia State, Brazil.

Introduction: In Brazil, an estimated 1.1 million people are infected with Trypanosoma cruzi, the causative agent of Chagas disease (CD). Despite the high number of cases, the estimated prevalence of infection per 100 inhabitants is low (0.03). However, the actual number of chronically infected individuals is still unknown. Therefore, we sought to determine the prevalence of chronic CD in at-risk individuals in Caraíbas (Bahia, Brazil) through active case finding. Methods: A total of 572 individuals living in rural or urban areas of Caraíbas were eligible for the study. A serum sample was collected from 226 individuals, and the diagnosis performed according to international guidelines. Results: The overall prevalence of anti-T. cruzi IgG was 4.42%. The median age of anti-T. cruzi IgG-positive individuals was 54.5 years, and the female-to-male ratio was 1.5:1. The prevalence of anti-T. cruzi IgG was similar in rural (4.29%) and urban areas (4.65%). Discussion: Compared with national estimates, we concluded that Caraíbas had a high prevalence for chronic CD and a high risk for persistent transmission. Through our study, it was possible to monitor individuals who were unaware of their clinical condition, thus improving their quality of life.

Seroepidemiological Survey of Chronic Chagas Disease in a Rural Community in Southern Bahia, Brazil, Using Recombinant Chimeric Antigens.

Chagas disease (CD), caused by the parasite Trypanosoma cruzi, is a neglected tropical disease with life-threatening implications. In this study, we conducted a seroepidemiological survey to determine the prevalence and clinical profiles of CD in 217 individuals from an impoverished rural community in Southern Bahia, Brazil. The overall prevalence of CD in the studied community was 0.92%, detected through latent class analysis (LCA). Two individuals tested positive for anti-T. cruzi IgG, both being male farmers. One case was a 22-year-old man born in Camamu, with no evidence of congenital transmission, suggesting other routes of transmission such as vector-borne transmission due to migratory activities. The other case was a 69-year-old man born in São Felipe, who had lived in an adobe/brick house and had a pacemaker due to cardiac involvement caused by CD. The prevalence in this community was lower than expected, given the socioeconomic conditions and environmental factors that contribute to T. cruzi transmission. This could be attributed to the implementation of preventive measures and vector control programs by the Brazilian Government. However, continuous monitoring and surveillance are essential to sustain control efforts and detect any potential re-emergence of the disease. While the overall prevalence was low, the detection of positive cases underscores the need for continued surveillance and control measures in vulnerable populations, such as rural communities. Active surveillance, early diagnosis, and timely treatment are crucial in preventing disease progression and complications, thereby enhancing the effectiveness of screening and treatment programs.

Biomarkers for the diagnosis, treatment follow-up, and prediction of cardiac complications in Chagas disease in chronic phase: Recent advances.

Chagas disease is caused by the Trypanosoma cruzi parasite and is transmitted by infected triatomine bugs. This infection affects approximately 8 million people in the Americas, and due to globalisation and displacement, it is becoming increasingly common to find infected patients worldwide. Diagnosis of the disease in its acute form is relatively simple, as the parasite can be detected in peripheral blood smears, and symptoms are visible. However, in its chronic condition, the parasite is almost undetectable, and indirect tests are necessary to determine the presence of antibodies in infected patients. It is important to note that a single test is not enough to confirm the disease in this phase, as a second serological test should confirm the diagnosis. If the results are contradictory, a third test should be performed to confirm or discard the disease. Unfortunately, laboratories may not have access to all necessary tests in many rural areas where the disease is more frequent. Rapid tests to diagnose this disease present problems, such as significant variations in sensitivity and specificity in different countries. Therefore, searching for new biomarkers that allow for optimal correlation is essential. In this work, we have searched scientific literature from the last 10 years for mentions of novel biomarkers for diagnosis, treatment follow-up, and prediction of cardiac complications in Chagas disease in its chronic phase.

Activation-induced marker assays for identification of Trypanosoma cruzi-specific CD4 or CD8 T cells in chronic Chagas disease patients.

Antigen-specific T cells are central to the adaptive immune response against T. cruzi infection and underpin the efficacy of on-going vaccine strategies. In this context, the present study focuses on T-cell assays that define the parasite-specificity on the basis of upregulation of TCR stimulation-induced surface markers. For this purpose, we tested different dual marker combinations (OX40, CD25, CD40L, CD137, CD69, PD-L1, CD11a, CD49d, HLA-DR, CD38) to reliably identify activated CD4+ and CD8+ T-cell populations from PBMCs of chronic Chagas disease (CCD) patients after 12 or 24 h of stimulation with T. cruzi lysate. Results demonstrated that activation-induced markers (AIM) assays combining the expression of OX40, CD25, CD40L, CD137, CD69 and/or PD-L1 surface markers are efficient at detecting T. cruzi-specific CD4+ T cells in CCD patients, in comparison to non-infected donors, after both stimulation times. For CD8+ T cells, only PD-L1/OX40 after 24 h of antigen exposure resulted to be useful to track a parasite-specific response. We also demonstrated that the agnostic activation is mediated by different T. cruzi strains, such as Dm28c, CL Brener or Sylvio. Additionally, we successfully used this approach to identify the phenotype of activated T lymphocytes based on the expression of CD45RA and CCR7. Overall, our results show that different combinations of AIM markers represent an effective and simple tool for the detection of T. cruzi-specific CD4+ and CD8+ T cells.

Benznidazole, itraconazole, and their combination for the treatment of chronic experimental Chagas disease in dogs.

Treatment for Chagas disease has limited efficacy in the chronic phase. We evaluated benznidazole (BZ) and itraconazole (ITZ) individually and in association in dogs 16 months after infection with a BZ-resistant Trypanosoma cruzi strain. Four study groups (20 animals) were evaluated and treated for 60 days with BZ, ITZ, or BZ + ITZ, and maintained in parallel to control group infected and not treated (INT). All dogs were evaluated in the first, sixth, 12th, 18th and 24th months of study. Polymerase chain reaction (PCR) was negative in 2 of 3 animals in the BZ + ITZ group, 2 of 5 in the BZ group, and 4 of 5 in the ITZ group. Hemoculture performed in the 24th month was negative in all groups. Enzyme-linked immunoassay remained reactive in all treated animals. Echocardiography differentiated treated animals from control animals. Quantitative PCR analysis of cardiac tissue was negative in the BZ + ITZ and BZ groups, positive in 2 of 5 dogs in the ITZ group and in 2 of 3 dogs in the control group, but negative in colon tissue in all groups. Inflammation was significantly reduced in the right atrium and left ventricle of dogs treated with BZ + ITZ and BZ compared with those receiving ITZ alone. Fibrosis was absent in most dogs treated with BZ + ITZ, mild in those treated with BZ or ITZ alone, and intense in the control group. Parasitological and histopathological evaluations showed that BZ + ITZ treatment improved or stabilized the clinical condition of the dogs.

Isolation and molecular characterization of circulating extracellular vesicles from blood of chronic Chagas disease patients.

Extracellular vesicles (EVs) are lipid bilayer envelopes that encase several types of molecules. Their contents mostly reflect their cell origin and possible targets at other locations in the organism and can be modified in pathological conditions to interfere with intercellular communication, thus promoting disease establishment and development. These characteristics, in addition to their presence in virtually all body fluids, make such vesicles ideal for biomarker discovery in human diseases. Here, we describe the effect of different anticoagulants and the combination of two purification methods for isolation and characterization of circulating EVs from blood of chronic Chagas disease (CCD) patients. We illustrated this procedure by studying a population of patients with Chagas disease at the indeterminate chronic stage, in which the Trypanosoma cruzi is very scarce in circulation. EVs were harvested from blood collected without or with different anticoagulants. Protein and nanoparticle tracking analysis was used to measure EVs size and concentration. The EVs were purified by ultracentrifugation, followed by size-exclusion chromatography and characterized by chemiluminescent enzyme-linked immunosorbent assay and dot blot using antibodies that recognized parasite-derived EVs, such as hyperimmune sera, polyclonal and monoclonal antibodies against trans-sialidase and mucins. In parallel, antibodies against classical human EV markers CD9, CD63, CD81, and CD82, were also analyzed. The results showed that anticoagulants did not interfere with the analyzed parameters and circulating EVs from CCD patients contain T. cruzi antigens and classical human exosomal markers. Overall, our protocol is adequate for the isolation of the total circulating EVs and can serve as an important basis for further studies on biomarker discovery in Chagas' disease.

Impairment of Multiple Mitochondrial Energy Metabolism Pathways in the Heart of Chagas Disease Cardiomyopathy Patients.

Chagas disease cardiomyopathy (CCC) is an inflammatory dilated cardiomyopathy occurring in 30% of the 6 million infected with the protozoan Trypanosoma cruzi in Latin America. Survival is significantly lower in CCC than ischemic (IC) and idiopathic dilated cardiomyopathy (DCM). Previous studies disclosed a selective decrease in mitochondrial ATP synthase alpha expression and creatine kinase activity in CCC myocardium as compared to IDC and IC, as well as decreased in vivo myocardial ATP production. Aiming to identify additional constraints in energy metabolism specific to CCC, we performed a proteomic study in myocardial tissue samples from CCC, IC and DCM obtained at transplantation, in comparison with control myocardial tissue samples from organ donors. Left ventricle free wall myocardial samples were subject to two-dimensional electrophoresis with fluorescent labeling (2D-DIGE) and protein identification by mass spectrometry. We found altered expression of proteins related to mitochondrial energy metabolism, cardiac remodeling, and oxidative stress in the 3 patient groups. Pathways analysis of proteins differentially expressed in CCC disclosed mitochondrial dysfunction, fatty acid metabolism and transmembrane potential of mitochondria. CCC patients' myocardium displayed reduced expression of 22 mitochondrial proteins belonging to energy metabolism pathways, as compared to 17 in DCM and 3 in IC. Significantly, 6 beta-oxidation enzymes were reduced in CCC, while only 2 of them were down-regulated in DCM and 1 in IC. We also observed that the cytokine IFN-gamma, previously described with increased levels in CCC, reduces mitochondrial membrane potential in cardiomyocytes. Results suggest a major reduction of mitochondrial energy metabolism and mitochondrial dysfunction in CCC myocardium which may be in part linked to IFN-gamma. This may partially explain the worse prognosis of CCC as compared to DCM or IC.

Co-Exposure of Cardiomyocytes to IFN-γ and TNF-α Induces Mitochondrial Dysfunction and Nitro-Oxidative Stress: Implications for the Pathogenesis of Chronic Chagas Disease Cardiomyopathy.

Infection by the protozoan Trypanosoma cruzi causes Chagas disease cardiomyopathy (CCC) and can lead to arrhythmia, heart failure and death. Chagas disease affects 8 million people worldwide, and chronic production of the cytokines IFN-γ and TNF-α by T cells together with mitochondrial dysfunction are important players for the poor prognosis of the disease. Mitochondria occupy 40% of the cardiomyocytes volume and produce 95% of cellular ATP that sustain the life-long cycles of heart contraction. As IFN-γ and TNF-α have been described to affect mitochondrial function, we hypothesized that IFN-γ and TNF-α are involved in the myocardial mitochondrial dysfunction observed in CCC patients. In this study, we quantified markers of mitochondrial dysfunction and nitro-oxidative stress in CCC heart tissue and in IFN-γ/TNF-α-stimulated AC-16 human cardiomyocytes. We found that CCC myocardium displayed increased levels of nitro-oxidative stress and reduced mitochondrial DNA as compared with myocardial tissue from patients with dilated cardiomyopathy (DCM). IFN-γ/TNF-α treatment of AC-16 cardiomyocytes induced increased nitro-oxidative stress and decreased the mitochondrial membrane potential (ΔΨm). We found that the STAT1/NF-κB/NOS2 axis is involved in the IFN-γ/TNF-α-induced decrease of ΔΨm in AC-16 cardiomyocytes. Furthermore, treatment with mitochondria-sparing agonists of AMPK, NRF2 and SIRT1 rescues ΔΨm in IFN-γ/TNF-α-stimulated cells. Proteomic and gene expression analyses revealed that IFN-γ/TNF-α-treated cells corroborate mitochondrial dysfunction, transmembrane potential of mitochondria, altered fatty acid metabolism and cardiac necrosis/cell death. Functional assays conducted on Seahorse respirometer showed that cytokine-stimulated cells display decreased glycolytic and mitochondrial ATP production, dependency of fatty acid oxidation as well as increased proton leak and non-mitochondrial oxygen consumption. Together, our results suggest that IFN-γ and TNF-α cause direct damage to cardiomyocytes' mitochondria by promoting oxidative and nitrosative stress and impairing energy production pathways. We hypothesize that treatment with agonists of AMPK, NRF2 and SIRT1 might be an approach to ameliorate the progression of Chagas disease cardiomyopathy.

Immunomodulation and Antioxidant Activities as Possible Trypanocidal and Cardioprotective Mechanisms of Major Terpenes from Lippia alba Essential Oils in an Experimental Model of Chronic Chagas Disease.

In the late phase of Trypanosoma cruzi infection, parasite persistence and an exaggerated immune response accompanied by oxidative stress play a crucial role in the genesis of Chronic Chagasic Cardiomyopathy (CCC). Current treatments (Benznidazole (BNZ) and Nifurtimox) can effect only the elimination of the parasite, but are ineffective for late stage treatment and for preventing heart damage and disease progression. In vivo trypanocidal and cardioprotective activity has been reported for Lippia alba essential oils (EOs), ascribed to their two major terpenes, limonene and caryophyllene oxide. To investigate the role of antioxidant and immunomodulatory mechanisms behind these properties, chronic-T. cruzi-infected rats were treated with oral synergistic mixtures of the aforementioned EOs. For this purpose, the EOs were optimized through limonene-enrichment fractioning and by the addition of exogenous caryophyllene oxide (LIMOX) and used alone or in combined therapy with subtherapeutic doses of BNZ (LIMOXBNZ). Clinical, toxicity, inflammatory, oxidative, and parasitological (qPCR) parameters were assessed in cardiac tissue. These therapies demonstrated meaningful antioxidant and immunomodulatory activity on markers involved in CCC pathogenesis (IFN-γ, TNF-α, IL-4, IL-10, and iNOS), which could explain their significant trypanocidal properties and their noteworthy role in preventing, and even reversing, the progression of cardiac damage in chronic Chagas disease.

Clinical, electrocardiographic and echocardiographic evolution of chronic Chagas disease treated with nifurtimox on prolonged follow-up in Chile: observational study.

OBJECTIVES: This study aimed to describe the electrocardiographic and echocardiographic status of chronic Chagas disease (cChD) patients treated with nifurtimox. METHODS: An observational study was performed in 146 cChD patients followed over a mean of 7.9 years. RESULTS: Of the 146 patients, 41 (28.1%) with normal electrocardiogram (ECG) at baseline maintained this condition, 34 (23.3%) with altered ECG at baseline normalised the alterations, and 46 (31.5%) with ECG abnormalities at baseline maintained this condition [23 (15.8%) with small alterations]. Finally, 25 cases (17.1%) in indeterminate phase altered the ECG. Differences before and after follow-up (P < 0.001) were found. The percentage of beneficial treatment was different than expected by chance (Z = 4.8; P < 0.001) and the annual percentage of cases that developed ECG alterations was lower than that of a historical cohort of untreated patients (P < 0.001). An echocardiogram was performed in 68 patients with baseline ECG alterations. The ejection fraction (EF) was normal in 57 (83.8%) and abnormal in 11 (16.2%). In 38 patients with ECG abnormalities that did not progress after treatment, EF and segmental motility (SM) were normal in 31 (81.6%) and 26 (68.4%), respectively. In 17 patients with ECG abnormalities, EF and SM were normal in 15 (88.2%) and 14 (82.4%) cases, respectively. CONCLUSION: Less progression to cardiomyopathy compared with a historical untreated cohort as well as the EF/SM results in patients with abnormal ECG that did not progress and in indeterminate cChD that altered the ECG suggests a beneficial effect of nifurtimox.

Differential Expression of Immune Response Genes in Asymptomatic Chronic Chagas Disease Patients Versus Healthy Subjects.

Infection by the Trypanosoma cruzi parasite causes Chagas disease and triggers multiple immune mechanisms in the host to combat the pathogen. Chagas disease has a variable clinical presentation and progression, producing in the chronic phase a fragile balance between the host immune response and parasite replication that keeps patients in a clinically silent asymptomatic stage for years. Since the parasite is intracellular and replicates within cells, the cell-mediated response of the host adaptive immunity plays a critical role. This function is mainly orchestrated by T lymphocytes, which recognize parasite antigens and promote specific functions to control the infection. However, little is known about the immunological markers associated with this asymptomatic stage of the disease. In this large-scale analysis, the differential expression of 106 immune system-related genes has been analyzed using high-throughput qPCR in T. cruzi antigen-stimulated PBMC from chronic Chagas disease patients with indeterminate form (IND) and healthy donors (HD) from endemic and non-endemic areas of Chagas disease. This analysis revealed that there were no differences in the expression level of most genes under study between healthy donors from endemic and non-endemic areas determined by PCA and differential gene expression analysis. Instead, PCA revealed the existence of different expression profiles between IND patients and HD (p < 0.0001), dependent on the 32 genes included in PC1. Differential gene expression analysis also revealed 23 upregulated genes (expression fold change > 2) and 11 downregulated genes (expression fold change < 0.5) in IND patients versus HD. Enrichment analysis showed that several upregulated genes in IND patients participate in relevant immunological pathways such as antigen-dependent B cell activation, stress induction of HSP regulation, NO2-dependent IL12 pathway in NK cells, and cytokine-inflammatory response. The antigen-specific differential gene expression profile detected in these patients and the relevant immunological pathways that seem to be activated could represent potential biomarkers of the asymptomatic form of Chagas disease, helpful to diagnosis and infection control.

Trypanosoma cruzi Induces Regulatory B Cell Alterations in Patients With Chronic Chagas Disease.

The clinical evolution of patients with chronic Chagas disease (CCD) is mainly associated with an excessive inflammation and a defective immunomodulatory profile caused by the interaction between T. cruzi and the host. Regulatory B (Breg) cells exert immune suppression mostly through IL-10 production (B10 cells), but also through IL-10-independent mechanisms. Previously, we demonstrated that CCD patients with cardiomyopathy show changes in the ex vivo Breg cell phenotypic distribution although maintain IL-10 production capacity. Here, we sought to identify potential alterations on Breg cells upon in vitro stimulation. Isolated B cells from CCD patients with or without cardiomyopathy and non-infected (NI) donors were stimulated with T. cruzi lysate or CpG + CD40L, and characterized by flow cytometry based on the expression of CD24, CD27, CD38, and the regulatory molecules IL-10 and PD-L1. IL-10 and IL-17 secretion in the supernatant of B cells was evaluated by ELISA. Data showed that T. cruzi stimulation diminished the expression of CD24 and CD38 on CD27- B cells while reducing the percentage of CD24high inside CD27+ B cells. Furthermore, T. cruzi induced a regulatory B cell phenotype by increasing B10 cells and IL-10 secretion in all the groups. The innate-like B10 cells expansion observed in patients with cardiomyopathy would be associated with CD27- B10 cell subsets, while no predominant phenotype was found in the other groups. Patients with cardiomyopathy also displayed higher IL-17 secretion levels in T. cruzi-activated B cells. CpG + CD40L stimulation revealed that B cells from CCD patients and NI donors had the same ability to differentiate into B10 cells and secrete IL-10 in vitro. Additionally, CCD patients showed an increased frequency of CD24-CD27- B cells and a reduction in the percentage of CD24highCD27+ Breg cells, which appeared to be inversely correlated with the presence of T. cruzi DNA in blood. Finally, CCD patients exhibited a higher frequency of PD-L1+ B cells in T. cruzi-stimulated samples, suggesting that IL-10-independent mechanisms could also be tangled in the control of inflammation. Altogether, our results provide evidence about the potential role of Breg cells in the immune response developed against T. cruzi and its contribution to chronic Chagas cardiomyopathy.

Evaluation of 2 Lateral Flow Rapid Tests in the Diagnosis of Chagas Disease in the Washington Metropolitan Area.

We compared the accuracy of the Stat-Pak and Chagas Detect Plus with a latent class analysis. Sensitivity values of 89.7% and 91.9% and specificities of 97.1% and 80.3%, respectively, were seen in the serodiagnosis of Chagas disease in Hispanic immigrants, revealing the limitations of these tests in diverse populations.

Fenofibrate Increases the Population of Non-Classical Monocytes in Asymptomatic Chagas Disease Patients and Modulates Inflammatory Cytokines in PBMC.

Chronic Chagas disease cardiomyopathy (CCC) is the most important clinical manifestation of infection with Trypanosma cruzi (T. cruzi) due to its frequency and effects on morbidity and mortality. Peripheral blood mononuclear cells (PBMC) infiltrate the tissue and differentiate into inflammatory macrophages. Advances in pathophysiology show that myeloid cell subpopulations contribute to cardiac homeostasis, emerging as possible therapeutic targets. We previously demonstrated that fenofibrate, PPARα agonist, controls inflammation, prevents fibrosis and improves cardiac function in a murine infection model. In this work we investigated the spontaneous release of inflammatory cytokines and chemokines, changes in the frequencies of monocyte subsets, and fenofibrate effects on PBMC of seropositive patients with different clinical stages of Chagas disease. The results show that PBMC from Chagas disease patients display higher levels of IL-12, TGF-ß, IL-6, MCP1, and CCR2 than cells from uninfected individuals (HI), irrespectively of the clinical stage, asymptomatic (Asy) or with Chagas heart disease (CHD). Fenofibrate reduces the levels of pro-inflammatory mediators and CCR2 in both Asy and CHD patients. We found that CHD patients display a significantly higher percentage of classical monocytes in comparison with Asy patients and HI. Besides, Asy patients have a significantly higher percentage of non-classical monocytes than CHD patients or HI. However, no difference in the intermediate monocyte subpopulation was found between groups. Moreover, monocytes from Asy or CHD patients exhibit different responses upon stimulation in vitro with T. cruzi lysates and fenofibrate treatment. Stimulation with T. cruzi significantly increases the percentage of classical monocytes in the Asy group whereas the percentage of intermediate monocytes decreases. Besides, there are no changes in their frequencies in CHD or HI. Notably, stimulation with T. cruzi did not modify the frequency of the non-classical monocytes subpopulation in any of the groups studied. Moreover, fenofibrate treatment of T. cruzi-stimulated cells, increased the frequency of the non-classical subpopulation in Asy patients. Interestingly, fenofibrate restores CCR2 levels but does not modify HLA-DR expression in any groups. In conclusion, our results emphasize a potential role for fenofibrate as a modulator of monocyte subpopulations towards an anti-inflammatory and healing profile in different stages of chronic Chagas disease.

Use of a Latent Class Analysis in the Diagnosis of Chronic Chagas Disease in the Washington Metropolitan Area.

BACKGROUND: The diversity of individuals at risk for Trypanosoma cruzi infection in the United States poses challenges for diagnosis. We evaluated the diagnostic accuracy of Food and Drug Administration (FDA)-cleared tests in the Washington Metropolitan area (WMA). METHODS: In total, 1514 individuals were evaluated (1078 from Mexico, Central and northern South America [TcI-predominant areas], and 436 from southern South America [TcII/V/VI-predominant areas]). Optical density (OD) values from the Hemagen EIA and Chagatest v.3 Wiener, and categorical results of the IgG-TESA-blot (Western blot with trypomastigote excretory-secretory antigen), and the Chagas detect plus (CDP), as well as information of area of origin were used to determine T. cruzi serostatus using latent class analysis. RESULTS: We detected 2 latent class (LC) of seropositives with low (LC1) and high (LC2) antibody levels. A significantly lower number of seropositives were detected by the Wiener, IgG-TESA-blot, and CDP in LC1 (60.6%, P < .001, 93.1%, P = .014, and 84.9%, P = .002, respectively) as compared to LC2 (100%, 100%, and 98.2%, respectively). LC1 was the main type of seropositives in TcI-predominant areas, representing 65.0% of all seropositives as opposed to 22.8% in TcII/V/VI-predominant areas. The highest sensitivity was observed for the Hemagen (100%, 95% confidence interval [CI]: 96.2-100.0), but this test has a low specificity (90.4%, 95% CI: 88.7-91.9). The best balance between positive (90.9%, 95% CI: 83.5-95.1), and negative (99.9%, 95% CI: 99.4-99.9) predictive values was obtained with the Wiener. CONCLUSIONS: Deficiencies in current FDA-cleared assays were observed. Low antibody levels are the main type of seropositives in individuals from TcI-predominant areas, the most frequent immigrant group in the United States.

Co-exposure of cardiomyocytes to IFN-γ and TNF-α induces mitochondrial dysfunction and nitro-oxidative stress: implications for the pathogenesis of chronic chagas disease cardiomyopathy

Infection by the protozoan Trypanosoma cruzi causes Chagas disease cardiomyopathy (CCC) and can lead to arrhythmia, heart failure and death. Chagas disease affects 8 million people worldwide, and chronic production of the cytokines IFN-γ and TNF-α by T cells together with mitochondrial dysfunction are important players for the poor prognosis of the disease. Mitochondria occupy 40% of the cardiomyocytes volume and produce 95% of cellular ATP that sustain the life-long cycles of heart contraction. As IFN-γ and TNF-α have been described to affect mitochondrial function, we hypothesized that IFN-γ and TNF-α are involved in the myocardial mitochondrial dysfunction observed in CCC patients. In this study, we quantified markers of mitochondrial dysfunction and nitro-oxidative stress in CCC heart tissue and in IFN-γ/TNF-α-stimulated AC-16 human cardiomyocytes. We found that CCC myocardium displayed increased levels of nitro-oxidative stress and reduced mitochondrial DNA as compared with myocardial tissue from patients with dilated cardiomyopathy (DCM). IFN-γ/TNF-α treatment of AC-16 cardiomyocytes induced increased nitro-oxidative stress and decreased the mitochondrial membrane potential (ΔΨm). We found that the STAT1/NF-κB/NOS2 axis is involved in the IFN-γ/TNF-α-induced decrease of ΔΨm in AC-16 cardiomyocytes. Furthermore, treatment with mitochondria-sparing agonists of AMPK, NRF2 and SIRT1 rescues ΔΨm in IFN-γ/TNF-α-stimulated cells. Proteomic and gene expression analyses revealed that IFN-γ/TNF-α-treated cells corroborate mitochondrial dysfunction, transmembrane potential of mitochondria, altered fatty acid metabolism and cardiac necrosis/cell death. Functional assays conducted on Seahorse respirometer showed that cytokine-stimulated cells display decreased glycolytic and mitochondrial ATP production, dependency of fatty acid oxidation as well as increased proton leak and non-mitochondrial oxygen consumption. Together, our results suggest that IFN-γ and TNF-α cause direct damage to cardiomyocytes’ mitochondria by promoting oxidative and nitrosative stress and impairing energy production pathways. We hypothesize that treatment with agonists of AMPK, NRF2 and SIRT1 might be an approach to ameliorate the progression of Chagas disease cardiomyopathy.

Clinical significance of tissue Doppler imaging in chronic Chagas disease.

INTRODUCTION: Chagas disease (ChD) is one of the main parasitic diseases in Latin-America. Its heart involvement is the most important cause of death. The aim of this study is to evaluate if Doppler Tissue Imaging (DTI) may have a predictive value for later events in subjects with chronic ChD. METHODS: we analyses DTI variables of 543 patients with chronic ChD for the evaluation of predicting factors of events. Major adverse cardiovascular events (MACE) were considered as stroke, heart failure resistant to treatment, sustained ventricular tachycardia, implantable cardioverter-defibrillator, sudden death, and cardiovascular death. The following findings were also included in total evens: heart failure, bradycardia, ventricular arrhythmia, new conduction system abnormalities, and new echocardiographic abnormalities. Multivariate analysis with logistic regression was used in order to assess the Doppler and DTI parameters predicting events. Variables with a P-value ≤ .5 in the univariate analysis were included in the multivariate analysis. RESULTS: In patients with chronic ChD, the analysis of DTI parameters showed that S' wave and E' wave of the lateral wall of the left ventricle were significant predictors of MACE (OR: 0.83; 95% CI: 0.71-0.96; P-value: .015 and OR: 0.80; 95% CI: 0.66-0.98; P-value: .031, respectively). CONCLUSIONS: This study found that patients with chronic ChD who had events showed significantly lower parameters in the DTI. What is more, this study showed that even lower DTI parameters are significant predictors of events.