TGF-β involvement in Chagas disease cardiomyopathy has been clearly demonstrated. The TGF-β signaling pathway is activated in the cardiac tissue of chronic phase patients and is associated with an increase in extracellular matrix protein expression. The aim of this study was to investigate the effect of GW788388, a selective inhibitor of TβR1/ALK5, on cardiac function in an experimental model of chronic Chagas’ heart disease. To this end, C57BL/6 mice were infected with Trypanosoma cruzi (10 2 parasites from the Colombian strain) and treated orally with 3mg/kg GW788388 starting at 120 days post-infection (dpi), when 100% of the infected mice show cardiac damage, and following three distinct treatment schedules: i) single dose; ii) one dose per week; or iii) three doses per week during 30 days. The treatment with GW788388 improved several cardiac parameters: reduced the prolonged PR and QTc intervals, increased heart rate, and reversed sinus arrhythmia, and atrial and atrioventricular conduction disorders. At 180 dpi, 30 days after treatment interruption, the GW3x-treated group remained in a better cardiac functional condition. Further, GW788388 treatment reversed the loss of connexin-43 enriched intercellular plaques and reduced fibrosis of the cardiac tissue. Inhibition of the TGF-β signaling pathway reduced TGF-β/pSmad2/3, increased MMP-9 and Sca-1, reduced TIMP-1/TIMP-2/TIMP-4, and partially restored GATA-6 and Tbox-5 transcription, supporting cardiac recovery. Moreover, GW788388 administration did not modify cardiac parasite load during the infection but reduced the migration of CD3 + cells to the heart tissue. Altogether, our data suggested that the single dose schedule was not as effective as the others and treatment three times per week during 30 days seems to be the most effective strategy. The therapeutic effects of GW788388 are promising and suggest a new possibility to treat cardiac fibrosis in the chronic phase of Chagas’ heart disease by TGF-β inhibitors.

The aim of the present study was to assess the effects of an anticholinesterase agent, pyridostigmine bromide (Pyrido), on experimental chronic Chagas heart disease in mice. To this end, male C57BL/6J mice noninfected (control:Con) or chronically infected (5 months) with Trypanosoma cruzi (chagasic:Chg) were treated or not (NT) with Pyrido for one month. At the end of this period, electrocardiogram (ECG); cardiac autonomic function; heart histopathology; serum cytokines; and the presence of blood and tissue parasites by means of immunohistochemistry and PCR were assessed. In NT-Chg mice, significant changes in the electrocardiographic, autonomic, and cardiac histopathological profiles were observed confirming a chronic inflammatory response. Treatment with Pyrido in Chagasic mice caused a significant reduction of myocardial inflammatory infiltration, fibrosis, and hypertrophy, which was accompanied by a decrease in serum levels of IFNγ with no change in IL-10 levels, suggesting a shift of immune response toward an anti-inflammatory profile. Lower nondifferent numbers of parasite DNA copies were observed in both treated and nontreated chagasic mice. In conclusion, our findings confirm the marked neuroimmunomodulatory role played by the parasympathetic autonomic nervous system in the evolution of the inflammatory-immune response to T. cruzi during experimental chronic Chagas heart disease in mice.

Background The protozoan Trypanosoma cruzi is auxotrophic for purines and causes Chagas’ disease (CD), a neglected illness affecting >6 million people. Combining the 3-deoxyribofuranose part of cordycepin with the modified purine ring of a nucleoside ‘hit’ led to the discovery of 4-amino-5-(4-chlorophenyl)-N7-(3′-deoxy-β-d-ribofuranosyl)-pyrrolo[2,3-d]pyrimidine (Cpd1), revealing promising anti-T. cruzi activity. Objectives To further evaluate Cpd1 in vitro and in vivo to fully assess its therapeutic potential against CD, covering cell culture sterilization through washout assays, drug combination with benznidazole and long-term administration in T. cruzi-infected mice. Results Although less susceptible to Cpd1 than amastigotes, trypomastigotes present an impaired capacity to successfully establish intracellular infection of cardiac cultures. Combination of benznidazole with Cpd1 indicated no interaction (additive effect) (FIC index = 0.72) while administration to mice at one-tenth of the optimal dose (2.5 mg/kg and 10 mg/kg for Cpd1 and benznidazole, respectively) suppressed parasitaemia but failed to avoid mortality. Long-term treatment (60 days) gave a rapid drop of the parasitaemia (>98% decline) and 100% mice survival but only 16% cure. In vitro washout experiments demonstrated that although parasite release into the supernatant of infected cardiac cultures was reduced by >94%, parasite recrudescence did occur after treatment. Conclusions Parasite recrudescence did occur after treatment corroborating the hypothesis of therapeutic failure due to subpopulations of dormant forms and/or genetic factors in persister parasites involved in natural drug resistance.

Background: Chagas disease (CD) has high morbimortality and the available trypanocidal treatment, including benznidazole (BZ), has limited efficacy in chronic patients. Furthermore, BZ causes adverse effects (AE) that prevent treatment completion in up to 30% of patients. The use of repositioned drugs or drug combination may provide an effective trypanocidal treatment. Disulfiram (DF) may enhance BZ activity and decrease BZ related AE. This study aims to assess the safety of a new combination of drugs for CD therapy, assuming BZ as the drug of choice plus DF as repositioned drug. Methods: This single-centre, open-label, phase I/II clinical trial was designed to evaluate the safety of the combined use of BZ plus DF for CD therapy. Participants are adults with indeterminate form of chronic CD, both sexes, aged from 18 to 70 years old and Trypanosoma cruzi polymerase chain reaction-positive. The primary outcome will be the occurrence of serious AE. The secondary outcome will be post-treatment Trypanosoma cruzi polymerase chain reaction negativization. Six groups of 9 patients will be sequentially tested. The first group will be allocated to receive BZ 100 mg/d + DF 250 mg/d for 60 days. Upon safety confirmation (<1/3 of participants with serious AE), the combination dose will be gradually increased and dispensed to 5 groups (group II:BZ 200 mg/day+DF 250 mg/d; group III:BZ 300 mg/d + DF 250 mg/d; group IV:BZ 100 mg/d + DF 500 mg/d; group V:BZ 200 mg/d + DF 500 mg/d; group VI: BZ 300 mg/d + DF 500 mg/d) for 60 days in order to determine the maximum tolerated dose. Discussion: Our hypothesis is that the drug combination will be well tolerated and allow the proposal of phase II trials in larger scale to test the efficacy of the new drug combination in CD. We expect that the studied combination will have less AEs with an efficacy similar or superior to the current treatment. This will allow the successful treatment of a greater number of patients while decreasing the treatment cost as less patients will need treatment for AEs. Trial registration: This study was registered on the Brazilian Clinical Trials Database - REBEC (RBR-5n4htp). Registered 7 January 2020. UTN Number: U1111-1246-1293. http://www.ensaiosclinicos.gov.br/rg/RBR-5n4htp/

In the absence of validated biomarkers to control the cure of Chagas disease, PCR-based diagnosis is being used as the main tool for an early indication of therapeutic failure. However, since it is considered a technique of complex reproducibility, mainly due to difficulties in establishing accurate controls to guarantee the quality of the reaction, the use of PCR for Chagas disease diagnosis is restricted to specialized centers. In an effort to disseminate the molecular diagnosis of Chagas disease and its applications, new diagnostic kits based on qPCR have been made available in the market in recent years. Here, we show the results of the validation of the NAT Chagas kit (Nucleic Acid Test for Chagas Disease) for the detection and quantification of T. cruzi in blood samples of patients suspected of Chagas disease infection. The kit, composed of a TaqMan duplex reaction targeting the T. cruzi satellite nuclear DNA and an exogenous internal amplification control, presented a reportable range from 104 to 0.5 parasite equivalents/mL and a limit of detection (LOD) of 0.16 parasite equivalents/mL of blood. In addition, the NAT Chagas kit detected T. cruzi belonging to all six discrete typing units (DTUs—TcI to TcVI), similarly to the in-house real-time PCR performed with commercial reagents, which has been selected as the best performance assay in the international consensus for the validation of qPCR for Chagas disease. In the clinical validation presented here, the kit showed 100% sensitivity and 100% specificity when compared to the consensus in-house real-time PCR assay. Thus, the NAT Chagas kit, which is produced entirely in Brazil under the international standards of good manufacturing practices (GMP), appears as an excellent alternative to enable the molecular diagnosis of Chagas disease in public and private diagnostic centers, as well as to facilitate the monitoring of patients under etiological treatment participating in clinical trials.