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.

Benznidazole modulates release of inflammatory mediators by cardiac spheroids infected with Trypanosoma cruzi.

Chagas disease (CD) caused by Trypanosoma cruzi remains a serious public health problem in Latin America. The available treatment is limited to two old drugs, benznidazole (Bz) and nifurtimox, which exhibit limited efficacy and trigger side effects, justifying the search for new therapies. Also, more accurate and sensitive experimental protocols for drug discovery programs are necessary to shrink the translational gaps found among pre-clinical and clinical trials. Presently, cardiac spheroids were used to evaluate host cell cytotoxicity and anti-T.cruzi activity of benznidazole, exploring its effect on the release of inflammatory mediators. Bz presented low toxic profile on 3D matrices (LC50 > 200 µM) and high potency in vitro (EC50 = 0.99 µM) evidenced by qPCR analysis of T.cruzi-infected cardiac spheroids. Flow cytometry appraisal of inflammatory mediators released at the cellular supernatant showed increases in IL - 6 and TNF contents (≈190 and ≈ 25-fold) in parasitized spheroids as compared to uninfected cultures. Bz at 10 µM suppressed parasite load (92%) concomitantly decreasing in IL-6 (36%) and TNF (68%). Our findings corroborate the successful use of 3D cardiac matrices for in vitro identification of novel anti-parasitic agents and potential impact in host cell physiology.

Innate immune receptors over expression correlate with chronic chagasic cardiomyopathy and digestive damage in patients.

Chronic chagasic cardiomyopathy (CCC) is observed in 30% to 50% of the individuals infected by Trypanosoma cruzi and heart failure is the important cause of death among patients in the chronic phase of Chagas disease. Although some studies have elucidated the role of adaptive immune responses involving T and B lymphocytes in cardiac pathogenesis, the role of innate immunity receptors such as Toll-like receptors (TLRs) and Nod-like receptors (NLRs) in CCC pathophysiology has not yet been determined. In this study, we evaluated the association among innate immune receptors (TLR1-9 and nucleotide-binding domain-like receptor protein 3/NLRP3), its adapter molecules (Myd88, TRIF, ASC and caspase-1) and cytokines (IL-1ß, IL-6, IL-12, IL-18, IL-23, TNF-α, and IFN-ß) with clinical manifestation, digestive and cardiac function in patients with different clinical forms of chronic Chagas disease. The TLR8 mRNA expression levels were enhanced in the peripheral blood mononuclear cells (PBMC) from digestive and cardiodigestive patients compared to indeterminate and cardiac patients. Furthermore, mRNA expression of IFN-ß (cytokine produced after TLR8 activation) was higher in digestive and cardiodigestive patients when compared to indeterminate. Moreover, there was a positive correlation between TLR8 and IFN-ß mRNA expression with sigmoid and rectum size. Cardiac and cardiodigestive patients presented higher TLR2, IL-12 and TNF-α mRNA expression than indeterminate and digestive patients. Moreover, cardiac patients also expressed higher levels of NLRP3, ASC and IL-1ß mRNAs than indeterminate patients. In addition, we showed a negative correlation among TLR2, IL-1ß, IL-12 and TNF-α levels with left ventricular ejection fraction, and positive correlation between NLRP3 with cardiothoracic index, and TLR2, IL-1ß and IL-12 with left ventricular mass index. Together, our data suggest that high expression of innate immune receptors in cardiac and digestive patients may induce an enhancement of cytokine expression and participate of cardiac and digestive dysfunction.

Virus-like Particle Display of the α-Gal Epitope for the Diagnostic Assessment of Chagas Disease.

The α-Gal antigen [Galα(1,3)Galß(1,4)GlcNAcα] is an immunodominant epitope displayed by infective trypomastigote forms of Trypanosoma cruzi, the causative agent of Chagas disease. A virus-like particle displaying a high density of α-Gal was found to be a superior reagent for the ELISA-based serological diagnosis of Chagas disease and the assessment of treatment effectiveness. A panel of sera from patients chronically infected with T. cruzi, both untreated and benznidazole-treated, was compared with sera from patients with leishmaniasis and from healthy donors. The nanoparticle-α-Gal construct allowed for perfect discrimination between Chagas patients and the others, avoiding false negative and false positive results obtained with current state-of-the-art reagents. As previously reported with purified α-Gal-containing glycosylphosphatidylinositol-anchored mucins, the current study also showed concentrations of anti-α-Gal IgG to decrease substantially in patients receiving treatment with benznidazole, suggesting that the semiquantitative assessment of serum levels of this highly abundant type of antibody can report on disease status in individual patients.

Characteristics of Triatomine infestation and natural Trypanosoma cruzi infection in the State of Rio Grande do Norte, Brazil.

INTRODUCTION Natural and artificial ecotope infestation by the kissing bug triatomines and their colonization and infection by Trypanosoma cruzi , the Chagas disease agent, were evaluated in nine municipalities of the State of Rio Grande do Norte, Brazil. METHODS Following identification, triatomine intestinal contents were analyzed by direct microscopic examination, xenoculture, and polymerase chain reaction (PCR) for parasite detection. Trypanosoma cruzi isolates were genotyped using three different markers. RESULTS Of 842 triatomines captured, 65% were Triatoma brasiliensis , 17.8% Triatoma pseudomaculata , 12.5% Panstrongylus lutzi , and 4.7% Rhodnius nasutus . Triatoma brasiliensis and P. lutzi adults were found in the intradomicile. T. brasiliensis, T. pseudomaculata , and R. nasutus nymphs and adults were found in the peridomicile and wild environment. Intradomiciliary and peridomiciliary infestation indexes were 5.6% and 33.7%, respectively. In the peridomicile, chicken coops were the most infested ecotope. The T. cruzi triatomine infection rate was 30.2%, of which PCR detected 29%. P . lutzi (78.1%), T . brasiliensis (24.5%), and T . pseudomaculata (22.7%) were the most infected species. TcII and III genotypes were detected in T. brasiliensis and TcIII in P. lutzi . CONCLUSIONS T. brasiliensis was found in all environments and most ecotopes with high T. cruzi infection rates. High infection rates were also detected in T . pseudomaculata and P. lutzi , suggesting their role in the interchange between the wild and peridomestic transmission cycles. The combination of PCR, microscopic examination, and xenoculture contributed to improving T. cruzi infection evaluation in triatomine bugs. The TcII and TcIII genotypes were predominant in the study area.

Inflammation Enhances the Risks of Stroke and Death in Chronic Chagas Disease Patients.

Ischemic strokes have been implicated as a cause of death in Chagas disease patients. Inflammation has been recognized as a key component in all ischemic processes, including the intravascular events triggered by vessel interruption, brain damage and repair. In this study, we evaluated the association between inflammatory markers and the death risk (DR) and stroke risk (SR) of patients with different clinical forms of chronic Chagas disease. The mRNA expression levels of cytokines, transcription factors expressed in the adaptive immune response (Th1, Th2, Th9, Th17, Th22 and regulatory T cell), and iNOS were analyzed by real-time PCR in peripheral blood mononuclear cells of chagasic patients who exhibited the indeterminate, cardiac, digestive and cardiodigestive clinical forms of the disease, and the levels of these transcripts were correlated with the DR and SR. Cardiac patients exhibited lower mRNA expression levels of GATA-3, FoxP3, AHR, IL-4, IL-9, IL-10 and IL-22 but exhibited higher expression of IFN-γ and TNF-α compared with indeterminate patients. Digestive patients showed similar levels of GATA-3, IL-4 and IL-10 than indeterminate patients. Cardiodigestive patients exhibited higher levels of TNF-α compared with indeterminate and digestive patients. Furthermore, we demonstrated that patients with high DR and SR exhibited lower GATA-3, FoxP3, and IL-10 expression and higher IFN-γ, TNF-α and iNOS mRNA expression than patients with low DR and SR. A negative correlation was observed between Foxp3 and IL-10 mRNA expression and the DR and SR. Moreover, TNF-α and iNOS expression was positively correlated with DR and SR. Our data suggest that an inflammatory imbalance in chronic Chagas disease patients is associated with a high DR and SR. This study provides a better understanding of the stroke pathobiology in the general population and might aid the development of therapeutic strategies for controlling the morbidity and mortality of Chagas disease.

Characteristics of Triatomine infestation and natural Trypanosoma cruzi infection in the State of Rio Grande do Norte, Brazil

Abstract: INTRODUCTION Natural and artificial ecotope infestation by the kissing bug triatomines and their colonization and infection by Trypanosoma cruzi , the Chagas disease agent, were evaluated in nine municipalities of the State of Rio Grande do Norte, Brazil. METHODS Following identification, triatomine intestinal contents were analyzed by direct microscopic examination, xenoculture, and polymerase chain reaction (PCR) for parasite detection. Trypanosoma cruzi isolates were genotyped using three different markers. RESULTS Of 842 triatomines captured, 65% were Triatoma brasiliensis , 17.8% Triatoma pseudomaculata , 12.5% Panstrongylus lutzi , and 4.7% Rhodnius nasutus . Triatoma brasiliensis and P. lutzi adults were found in the intradomicile. T. brasiliensis, T. pseudomaculata , and R. nasutus nymphs and adults were found in the peridomicile and wild environment. Intradomiciliary and peridomiciliary infestation indexes were 5.6% and 33.7%, respectively. In the peridomicile, chicken coops were the most infested ecotope. The T. cruzi triatomine infection rate was 30.2%, of which PCR detected 29%. P . lutzi (78.1%), T . brasiliensis (24.5%), and T . pseudomaculata (22.7%) were the most infected species. TcII and III genotypes were detected in T. brasiliensis and TcIII in P. lutzi . CONCLUSIONS T. brasiliensis was found in all environments and most ecotopes with high T. cruzi infection rates. High infection rates were also detected in T . pseudomaculata and P. lutzi , suggesting their role in the interchange between the wild and peridomestic transmission cycles. The combination of PCR, microscopic examination, and xenoculture contributed to improving T. cruzi infection evaluation in triatomine bugs. The TcII and TcIII genotypes were predominant in the study area.

Chagas disease: morbidity profile in an endemic area of Northeastern Brazil.

INTRODUCTION: This study evaluated the clinical forms and manifestation severities of Chagas disease among serologically reactive individuals from Western Rio Grande do Norte (Northeastern Brazil). METHODS: This cross-sectional study included 186 adults who were evaluated using electrocardiography, echocardiography, chest radiography, and contrast radiography of the esophagus and colon. A clinical-epidemiological questionnaire was also used. RESULTS: The indeterminate, cardiac, digestive, and cardiodigestive clinical forms of Chagas disease were diagnosed in 51.6% (96/186), 32.2% (60/186), 8.1% (15/186) and 8.1% (15/186) of the participants, respectively. Heart failure (functional classes I-IV) was detected in 7.5% (14/186) of the participants, and 36.4% (24/66), 30.3% (20/66), 15.2% (10/66), 13.6% (9/66), and 4.5% (3/66) of the patients were at stage A, B1, B2, C, and D, respectively. Dilated cardiomyopathy and electrocardiographic changes were detected in 10.2% (19/186) and 48.1% (91/186) of the participants, respectively. Apical aneurysm was diagnosed in 10.8% (20/186) of the participants, and other changes in the segmental myocardial contractility of the left ventricle were diagnosed in 33.9% (63/186) of the participants. Megaesophagus (groups I-IV) was observed in 7% (13/186) of the participants, megacolon (grades 1-3) was detected in 12.9% (24/186) of the participants, and both organs were affected in 29.2% (7/24) of the megacolon cases. CONCLUSIONS: We detected various clinical forms of Chagas disease (including the digestive form). Our findings indicate that clinical symptoms alone may not be sufficient to exclude or confirm cardiac and/or digestive damage, and the number of patients with symptomatic clinical forms may be underestimated.

Chagas disease: morbidity profile in an endemic area of Northeastern Brazil

Abstract: INTRODUCTION : This study evaluated the clinical forms and manifestation severities of Chagas disease among serologically reactive individuals from Western Rio Grande do Norte (Northeastern Brazil). METHODS : This cross-sectional study included 186 adults who were evaluated using electrocardiography, echocardiography, chest radiography, and contrast radiography of the esophagus and colon. A clinical-epidemiological questionnaire was also used. RESULTS : The indeterminate, cardiac, digestive, and cardiodigestive clinical forms of Chagas disease were diagnosed in 51.6% (96/186), 32.2% (60/186), 8.1% (15/186) and 8.1% (15/186) of the participants, respectively. Heart failure (functional classes I-IV) was detected in 7.5% (14/186) of the participants, and 36.4% (24/66), 30.3% (20/66), 15.2% (10/66), 13.6% (9/66), and 4.5% (3/66) of the patients were at stage A, B1, B2, C, and D, respectively. Dilated cardiomyopathy and electrocardiographic changes were detected in 10.2% (19/186) and 48.1% (91/186) of the participants, respectively. Apical aneurysm was diagnosed in 10.8% (20/186) of the participants, and other changes in the segmental myocardial contractility of the left ventricle were diagnosed in 33.9% (63/186) of the participants. Megaesophagus (groups I-IV) was observed in 7% (13/186) of the participants, megacolon (grades 1-3) was detected in12.9% (24/186) of the participants, and both organs were affected in 29.2% (7/24) of the megacolon cases. CONCLUSIONS : We detected various clinical forms of Chagas disease (including the digestive form). Our findings indicate that clinical symptoms alone may not be sufficient to exclude or confirm cardiac and/or digestive damage, and the number of patients with symptomatic clinical forms may be underestimated.

Troponin T autoantibodies correlate with chronic cardiomyopathy in human Chagas disease.

OBJECTIVE: To evaluate the potential involvement of anti-Trypanosoma cruzi and cardiac protein antibody (IgG total and isotypes) production and their possible association with different clinical forms of human chronic Chagas disease. METHODS: IgG total and isotypes were measured by ELISA, using epimastigote and trypomastigote forms of T. cruzi as antigens and human cardiac proteins (myosin and troponin T) in sera of patients with indeterminate (IND, n = 72), cardiac (CARD, n = 47) and digestive/cardiodigestive (DIG/CARD-DIG, n = 12) clinical forms of the disease. Samples from uninfected health individuals (CONT, n = 30) and patients with ischaemic cardiomyopathy (ISCH, n = 15) were used as controls. Autoantibody levels were correlated with parameters of cardiac function obtained by electrocardiographic, radiographic and echocardiographic examinations. RESULTS: Fifty five per cent of patients were classified as IND, 35.9% as CARD and 9.1% as DIG/CARD-DIG. Greater total IgG production was observed in IND, CARD and DIG/CARD-DIG chagasic patients than in CONT and ISCH, using trypomastigote, epimastigote and cardiac antigens. Moreover, patients with CARD and DIG/CARD-DIG presented greater total IgG production (trypomastigote and epimastigote antigen) than IND, and a negative correlation was determined between total IgG and left ventricular ejection fraction (LVEF). Patients with IND and CARD presented similar higher levels of total IgG specific to troponin T and myosin than CONT and ISCH individuals. Patients with chronic Chagas disease presented a negative correlation between left ventricular ejection fraction (LVEF) and the production of anti-myosin and troponin T autoantibodies. When grouped as low and high antibody producers and compared with LVEF, we observed that high anti-troponin T (P = 0.042) and myosin (P = 0.013) producers presented lower LVEF than low producers. Moreover, there was a positive correlation (r = 0.9508, P = 0.0001) between the production of troponin T and myosin autoantibodies. CONCLUSION: These findings indicate that increased production of anti-cardiac troponin T and myosin autoantibodies probably influences the left ventricular ejection fraction and could be related to chagasic cardiomyopathy.

Seroepidemiology of Trypanosoma cruzi infection in the semiarid rural zone of the State of Rio Grande do Norte, Brazil.

INTRODUCTION: A seroepidemiological survey was carried out to evaluate Trypanosoma cruzi infection in an endemic area of the State of Rio Grande do Norte, Brazil, involving rural residents. METHODS: Sixteen municipalities were randomly selected, 15 from the west mesoregion and one from the central, with an estimated population of 83,852 individuals. A total of 1,950 blood samples were collected in the west mesoregion and 390 in Caicó. Anti-T. cruzi antibodies were detected using the Chagatest® ELISA HAI-hemagglutination kits and indirect immunofluorescence. As sera presented indeterminate results, TESAcruzi® western blot was performed to confirm reactivity. RESULTS: An estimated seroprevalence of 6.5% was determined for the west mesoregion and 3.3% for Caicó. Seropositivity rises progressively with the age of individuals, up to 40 years in Caicó and up to 50 years in the west mesoregion. Only educational level and knowledge regarding the triatomine were associated with seropositivity. No seroreactive individuals under 18 years of age were identified. CONCLUSIONS: Infection by T. cruzi remains high and is concentrated in municipalities in the central western area of the west mesoregion; however, evidence suggests a decline in vector transmission in this mesoregion and in Caicó. Epidemiological variables appear not to influence seropositivity, with the exception of education and knowledge concerning the triatomine, among seroreactive individuals from the west mesoregion.

Seroepidemiology of Trypanosoma cruzi infection in the semiarid rural zone of the State of Rio Grande do Norte, Brazil / Soroepidemiologia da infecção pelo Trypanosoma cruzi na zona rural do semiárido do Estado do Rio Grande do Norte, Brasil

INTRODUCTION: A seroepidemiological survey was carried out to evaluate Trypanosoma cruzi infection in an endemic area of the State of Rio Grande do Norte, Brazil, involving rural residents. METHODS: Sixteen municipalities were randomly selected, 15 from the west mesoregion and one from the central, with an estimated population of 83,852 individuals. A total of 1,950 blood samples were collected in the west mesoregion and 390 in Caicó. Anti-T. cruzi antibodies were detected using the Chagatest® ELISA HAI-hemagglutination kits and indirect immunofluorescence. As sera presented indeterminate results, TESAcruzi® western blot was performed to confirm reactivity. RESULTS: An estimated seroprevalence of 6.5% was determined for the west mesoregion and 3.3% for Caicó. Seropositivity rises progressively with the age of individuals, up to 40 years in Caicó and up to 50 years in the west mesoregion. Only educational level and knowledge regarding the triatomine were associated with seropositivity. No seroreactive individuals under 18 years of age were identified. CONCLUSIONS: Infection by T. cruzi remains high and is concentrated in municipalities in the central western area of the west mesoregion; however, evidence suggests a decline in vector transmission in this mesoregion and in Caicó. Epidemiological variables appear not to influence seropositivity, with the exception of education and knowledge concerning the triatomine, among seroreactive individuals from the west mesoregion.

INTRODUÇÃO: A infecção pelo Trypanosoma cruzi foi avaliada em uma área endêmica do Estado do Rio Grande do Norte, Brasil, por inquérito soroepidemiológico amostral em moradores da zona rural. MÉTODOS: Dezesseis municípios foram sorteados, 15 da mesorregião oeste e um da central, com população estimada em 83.852 indivíduos. Foram coletadas 1.950 amostras de sangue no oeste e 390 em Caicó. A detecção de anticorpos anti-T. cruzi foi realizada usando os kits Chagatest® ELISA, HAI-hemaglutinação e a reação de imunofluorescência indireta. Nos soros com resultados indeterminados foi realizado o western blot TESAcruzi® para confirmação da reatividade. RESULTADOS: A estimativa da soroprevalência revelou 6,5% para a mesorregião oeste e 3,3% em Caicó. A soropositividade eleva-se progressivamente com a idade dos indivíduos até a quinta década de vida em Caicó e na sexta década na mesorregião oeste. Apenas o grau de escolaridade e o conhecimento do triatomíneo evidenciaram associação à soropositividade. Não foram identificados indivíduos sororreativos com idade inferior a 18 anos. CONCLUSÕES: A infecção pelo T. cruzi persiste mais elevada e concentrada em municípios da área central da mesorregião oeste, mas evidências sugerem o declínio da transmissão vetorial nessa mesorregião e em Caicó. As variáveis epidemiológicas parecem não exercer influência na soropositividade, à exceção da escolaridade e conhecimento do triatomíneo entre indivíduos sororreativos da mesorregião oeste.