The expression of immune response genes in patients with chronic Chagas disease is shifted toward the levels observed in healthy subjects as a result of treatment with Benznidazole.

Introduction: Chagas disease, caused by the Trypanosoma cruzi parasite infection, is a potentially life-threatening neglected tropical disease with a worldwide distribution. During the chronic phase of the disease, there exists a fragile balance between the host immune response and parasite replication that keeps patients in a clinically-silent asymptomatic stage for years or even decades. However, in 40% of patients, the disease progresses to clinical manifestations mainly affecting and compromising the cardiac system. Treatment is recommended in the chronic phase, although there are no early markers of its effectiveness. The aim of this study is to identify differential expression changes in genes involved in the immune response in antigen-restimulated PBMC from chronic patients with Chagas disease due to benznidazole treatment. Methods: Thus, high-throughput real-time qPCR analysis has been performed to simultaneously determine global changes in the expression of 106 genes involved in the immune response in asymptomatic (IND) and early cardiac manifestations (CCC I) Chagas disease patients pre- and post-treatment with benznidazole. Results and discussion: The results revealed that 7 out of the 106 analyzed genes were differentially expressed (4 up- and 3 downregulated) after treatment in IND patients and 15 out of 106 (3 up- and 12 downregulated) after treatment of early cardiac Chagas disease patients. Particularly in CCC I patients, regulation of the expression level of some of these genes towards a level similar to that of healthy subjects suggests a beneficial effect of treatment and supports recommendation of benznidazole administration to early cardiac Chagas disease patients. The data obtained also demonstrated that both in asymptomatic patients and in early cardiac chronic patients, after treatment with benznidazole there is a negative regulation of the proinflammatory and cytotoxic responses triggered as a consequence of T. cruzi infection and the persistence of the parasite. This downregulation of the immune response likely prevents marked tissue damage and healing in early cardiac patients, suggesting its positive effect in controlling the pathology.

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

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

A Case of Chagas Disease in South Dakota: Diagnosis and Treatment.

Chagas disease is a chronic, systemic parasitic infection caused by the protozoan Trypanosoma cruzi. The primary mode of transmission to humans is by the Reduviid insect, endemic to South America. Recent migration of the vector has led to increased cases in the southern United States and has prompted increased surveillance and blood donation screening. It is unusual to diagnose and treat individuals with Chagas disease in the northern United States. This case describes an immigrant female from El Salvador that was informed she had Chagas disease from a blood bank screening. Confirmation and treatment of the disease were performed by her South Dakota primary care provider thus demonstrating the importance of identifying Chagas disease in the immigrant population in regions where Chagas disease infection is uncommon.

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

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

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

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

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.

Targeting Trypanothione Metabolism in Trypanosomatids.

Infectious diseases caused by trypanosomatids, including African trypanosomiasis (sleeping sickness), Chagas disease, and different forms of leishmaniasis, are Neglected Tropical Diseases affecting millions of people worldwide, mainly in vulnerable territories of tropical and subtropical areas. In general, current treatments against these diseases are old-fashioned, showing adverse effects and loss of efficacy due to misuse or overuse, thus leading to the emergence of resistance. For these reasons, searching for new antitrypanosomatid drugs has become an urgent necessity, and different metabolic pathways have been studied as potential drug targets against these parasites. Considering that trypanosomatids possess a unique redox pathway based on the trypanothione molecule absent in the mammalian host, the key enzymes involved in trypanothione metabolism, trypanothione reductase and trypanothione synthetase, have been studied in detail as druggable targets. In this review, we summarize some of the recent findings on the molecules inhibiting these two essential enzymes for Trypanosoma and Leishmania viability.

A woman in her seventies with fever and convulsions. / En kvinne i 70-årene med feber og krampeanfall.

Background: Chagas encephalitis is a rare but severe manifestation of reactivation in patients with chronic Chagas disease. Case presentation: A woman in her seventies who was immunosuppressed after a heart transplant due to Chagas disease was admitted with convulsions, headache and visual disturbances. She developed fever, confusion and repeated convulsions. Pleocytosis was found in spinal fluid. Wet-mount microscopy of spinal fluid revealed motile Trypanosoma cruzi trypomastigotes, and multiple trypomastigotes were seen on a Giemsa-stained smear, confirming reactivation of Chagas disease with meningoencephalitis. Despite benznidazole treatment, she deteriorated, exhibiting pharyngeal paralysis, aphasia and increasing somnolence. Brain CT showed pathology consistent with Chagas encephalitis. Nifurtimox was given as an adjunctive treatment. After a week of treatment, the patient began to improve. She completed 60 days of benznidazole and had regained normal cognitive and neurological function on subsequent follow-up. She had no signs of myocarditis reactivation. Interpretation: Chronic Chagas disease is common among Latin American immigrants in Europe. Reactivation with myocarditis after a heart transplant is well known, while encephalitis is a rare manifestation. We report on a case of Chagas encephalitis in an immunosuppressed patient. Microscopy of parasites in spinal fluid revealed the diagnosis. The WHO provided antiparasitic medications, and despite a severe prognosis, the patient made a full recovery.

Tackling the challenges of human Chagas disease: A comprehensive review of treatment strategies in the chronic phase and emerging therapeutic approaches.

Chagas disease (CD), caused by the flagellated protozoan Trypanosoma cruzi (T. cruzi), affects approximately 7 million people worldwide and is endemic in Latin America, especially among socioeconomically disadvantaged populations. Since the 1960s, only two drugs have been commercially available for treating this illness: nifurtimox (NFX) and benznidazole (BZN). Although these drugs are effective in the acute phase (AP) of the disease, in which parasitemia is usually high, their cure rates in the chronic phase (CP) are low and often associated with several side effects. The CP is characterized by a subpatent parasitaemia and absence of clinical symptoms in the great majority of infected individuals. However, at least 30 % of the individuals will develop potentially lethal symptomatic forms, including cardiac and digestive manifestations. For such reason, in the CP the treatment is usually symptomatic and typically focuses on managing complications such as arrhythmias, heart failure, or digestive problems. Therefore, the need for new drugs or therapeutic approaches using BZN or NFX is extremely urgent. This review presents the main clinical trials, especially in the CP, which involve BZN and NFX in different treatment regimens. Additionally, other therapies using combinations of these drugs with other substances such as allopurinol, itraconazole, ravuconazole, ketoconazole, posaconazole and amiodarone are also reported. The importance of early diagnosis, especially in pediatric patients, is also discussed, emphasizing the need to identify the disease in its early stages to improve the chances of successful treatment.

Enteric nervous system regeneration and functional cure of experimental digestive Chagas disease with trypanocidal chemotherapy.

Digestive Chagas disease (DCD) is an enteric neuropathy caused by Trypanosoma cruzi infection. There is a lack of evidence on the mechanism of pathogenesis and rationales for treatment. We used a female C3H/HeN mouse model that recapitulates key clinical manifestations to study how infection dynamics shape DCD pathology and the impact of treatment with the front-line, anti-parasitic drug benznidazole. Curative treatment 6 weeks post-infection resulted in sustained recovery of gastrointestinal transit function, whereas treatment failure led to infection relapse and gradual return of DCD symptoms. Neuro/immune gene expression patterns shifted from chronic inflammation to a tissue repair profile after cure, accompanied by increased cellular proliferation, glial cell marker expression and recovery of neuronal density in the myenteric plexus. Delaying treatment until 24 weeks post-infection led to partial reversal of DCD, suggesting the accumulation of permanent tissue damage over the course of chronic infection. Our study shows that murine DCD pathogenesis is sustained by chronic T. cruzi infection and is not an inevitable consequence of acute stage denervation. The risk of irreversible enteric neuromuscular tissue damage and dysfunction developing highlights the importance of prompt diagnosis and treatment. These findings support the concept of treating asymptomatic, T. cruzi-infected individuals with benznidazole to prevent DCD development.

Development of Novel Peptidyl Nitriles Targeting Rhodesain and Falcipain-2 for the Treatment of Sleeping Sickness and Malaria.

In recent decades, neglected tropical diseases and poverty-related diseases have become a serious health problem worldwide. Among these pathologies, human African trypanosomiasis, and malaria present therapeutic problems due to the onset of resistance, toxicity problems and the limited spectrum of action. In this drug discovery process, rhodesain and falcipain-2, of Trypanosoma brucei rhodesiense and Plasmodium falciparum, are currently considered the most promising targets for the development of novel antitrypanosomal and antiplasmodial agents, respectively. Therefore, in our study we identified a novel lead-like compound, i.e., inhibitor 2b, which we proved to be active against both targets, with a Ki = 5.06 µM towards rhodesain and an IC50 = 40.43 µM against falcipain-2.

Positive clinical outcome using a modified dosing regimen of benznidazole in dogs at high risk for infection or acutely infected with Trypanosoma cruzi.

Trypanosoma cruzi infection in dogs can cause heart failure and sudden death with few treatment options available. A litter of 4 dogs living in a T cruzi endemic area were randomized to prophylaxis and nonprophylaxis groups as part of a study evaluating a modified benznidazole dosing regimen administered twice weekly to prevent T cruzi infection during a vector transmission season. The 2 dogs that received prophylaxis remained healthy without T cruzi infection or cardiac disease for >2 years. One dog that did not receive prophylaxis died unexpectedly with acute T cruzi-induced pancarditis, and the second dog tested positive for T cruzi and developed complex arrhythmias with markedly increased cardiac troponin I and improved with a higher benznidazole treatment dose. Although the small sample size precludes definitive conclusions, we describe the potential clinical benefit of prophylactic and early treatment with modified benznidazole dosing regimens for dogs with T cruzi infection.

Ascofuranone antibiotic is a promising trypanocidal drug for nagana.

Trypanosomosis is a disease complex which affects both humans and animals in sub-Saharan Africa, transmitted by the tsetse fly and distributed within the tsetse belt of Africa. But some trypanosome species, for example, Trypanosoma brucei evansi, T. vivax, T. theileri and T. b. equiperdum are endemic outside the tsetse belt of Africa transmitted by biting flies, for example, Tabanus and Stomoxys, or venereal transmission, respectively. Trypanocidal drugs remain the principal method of animal trypanosomosis control in most African countries. However, there is a growing concern that their effectiveness may be severely curtailed by widespread drug resistance. A minimum number of six male cattle calves were recruited for the study. They were randomly grouped into two (T. vivax and T. congolense groups) of three calves each. One calf per group served as a control while two calves were treatment group. They were inoculated with 105 cells/mL parasites in phosphate buffered solution (PBS) in 2 mL. When parasitaemia reached 1 × 107.8 cells/mL trypanosomes per mL in calves, treatment was instituted with 20 mL (25 mg/kg in 100 kg calf) ascofuranone (AF) for treatment calves, while the control ones were administered a placebo (20 mL PBS) intramuscularly. This study revealed that T. vivax was successfully cleared by AF but the T. congolense group was not cleared effectively.Contribution: There is an urgent need to develop new drugs which this study sought to address. It is suggested that the AF compound can be developed further to be a sanative drug for T. vivax in non-tsetse infested areas like South Americas.

Selenosugars targeting the infective stage of Trypanosoma brucei with high selectivity.

Earlier evidences showed that diglycosyl diselenides are active against the infective stage of African trypanosomes (top hits IC50 0.5 and 1.5 µM) but poorly selective (selectivity index <10). Here we extended the study to 33 new seleno-glycoconjugates with the aim to improve potency and selectivity. Three selenoglycosides and three glycosyl selenenylsulfides displayed IC50 against bloodstream Trypanosoma brucei in the sub-µM range (IC50 0.35-0.77 µM) and four of them showed an improved selectivity (selectivity index >38-folds vs. murine and human macrohages). For the glycosyl selenylsulfides, the anti-trypanosomal activity was not significantly influenced by the nature of the moiety attached to the sulfur atom. Except for a quinoline-, and to a minor extent a nitro-derivative, the most selective hits induced a rapid (within 60 min) and marked perturbation of the LMWT-redox homeostasis. The formation of selenenylsulfide glycoconjugates with free thiols has been identified as a potential mechanism involved in this process.

Identification of a potent and selective LAPTc inhibitor by RapidFire-Mass Spectrometry, with antichagasic activity.

BACKGROUND: Chagas disease is caused by the protozoan parasite Trypanosoma cruzi and leads to ~10,000 deaths each year. Nifurtimox and benznidazole are the only two drugs available but have significant adverse effects and limited efficacy. New chemotherapeutic agents are urgently required. Here we identified inhibitors of the acidic M17 leucyl-aminopeptidase from T. cruzi (LAPTc) that show promise as novel starting points for Chagas disease drug discovery. METHODOLOGY/PRINCIPAL FINDINGS: A RapidFire-MS screen with a protease-focused compound library identified novel LAPTc inhibitors. Twenty-eight hits were progressed to the dose-response studies, from which 12 molecules inhibited LAPTc with IC50 < 34 µM. Of these, compound 4 was the most potent hit and mode of inhibition studies indicate that compound 4 is a competitive LAPTc inhibitor, with Ki 0.27 µM. Compound 4 is selective with respect to human LAP3, showing a selectivity index of >500. Compound 4 exhibited sub-micromolar activity against intracellular T. cruzi amastigotes, and while the selectivity-window against the host cells was narrow, no toxicity was observed for un-infected HepG2 cells. In silico modelling of the LAPTc-compound 4 interaction is consistent with the competitive mode of inhibition. Molecular dynamics simulations reproduce the experimental binding strength (-8.95 kcal/mol), and indicate a binding mode based mainly on hydrophobic interactions with active site residues without metal cation coordination. CONCLUSIONS/SIGNIFICANCE: Our data indicates that these new LAPTc inhibitors should be considered for further development as antiparasitic agents for the treatment of Chagas disease.

Lead Optimization of the 5-Phenylpyrazolopyrimidinone NPD-2975 toward Compounds with Improved Antitrypanosomal Efficacy.

Human African trypanosomiasis (HAT) still faces few therapeutic options and emerging drug resistance, stressing an urgency for novel antitrypanosomal drug discovery. Here, we describe lead optimization efforts aiming at improving antitrypanosomal efficacy and better physicochemical properties based on our previously reported optimized hit NPD-2975 (pIC50 7.2). Systematic modification of the 5-phenylpyrazolopyrimidinone NPD-2975 led to the discovery of a R4-substituted analogue 31c (NPD-3519), showing higher in vitro potency (pIC50 7.8) against Trypanosoma brucei and significantly better metabolic stability. Further, in vivo pharmacokinetic evaluation of 31c and experiments in an acute T. brucei mouse model confirmed improved oral bioavailability and antitrypanosomal efficacy at 50 mg/kg with no apparent toxicity. With good physicochemical properties, low toxicity, improved pharmacokinetic features, and in vivo efficacy, 31c may serve as a promising candidate for future drug development for HAT.

Antitrypanosomal Chloronitrobenzamides.

Human African trypanosomiasis (HAT), a neglected tropical disease caused by Trypanosoma brucei gambiense (Tbg) or Trypanosoma brucei rhodesiense (Tbr), remains a significant public health concern with over 55 million people at risk of infection. Current treatments for HAT face the challenges of poor efficacy, drug resistance, and toxicity. This study presents the synthesis and evaluation of chloronitrobenzamides (CNBs) against Trypanosoma species, identifying previously reported compound 52 as a potent and selective orally bioavailable antitrypanosomal agent. 52 was well tolerated in vivo and demonstrated favorable oral pharmacokinetics, maintaining plasma concentrations surpassing the cellular EC50 for over 24 h and achieving peak brain concentrations exceeding 7 µM in rodents after single oral administration (50 mg/kg). Treatment with 52 significantly extended the lifespan of mice infected with Trypanosoma congolense and T. brucei rhodesiense. These results demonstrate that 52 is a strong antitrypanosomal lead with potential for developing treatments for both human and animal African trypanosomiasis.

Parasitological, Serological and Molecular Prevalence of Trypanosoma evansi among Arabian Camels (Camelus dromedaries) with Evaluation of Antitrypanosomal Drugs.

PURPOSE: This study was carried out to assess the prevalence of Trypanosoma evansi infection in naturally diseased Dromedary camels in Dammam, Eastern region of Saudi Arabia. The detection of Trypanosoma evansi was performed using the parasitological, serological, and molecular diagnosis and a comparison between such methods were analyzed. In addition, evaluation of therapeutic efficacy of selected antitrypanosomal drugs, cymelarsan and quinapyrmine (aquin-1.5), was trialed for treatment of diagnosed infected cases. METHODS: A total 350 randomly selected camels were evaluated using thin blood smear (TBS), RoTat1.2 PCR and CATT/T. evansi techniques. RESULTS: The total prevalence was 6.9%, 7.7%, and 32.8% by TBS, RoTat1.2 PCR and CATT/T. evansi techniques, respectively. Although PCR detect T. evansi in more samples than TBS, the agreement was good (K = 0.9). Among the CATT/T. evansi results, PCR detect T. evansi in 12 and 15 CATT positive and negative camels, respectively, with low agreement (Kappa = 0.1). The use of cymelarsan and quinapyramine sulfate in the treatment of naturally infected cases demonstrated a very efficient therapeutic response. CONCLUSION: It was found that 1. Comparing the CATT/T. evansi and PCR results, the positivity of CATT was higher than PCR detection, while the agreement was poor (K = 0.1). 2. Cymelarsan and aquin-1.5 proved to be effective in the treatment of naturally infected camels, but cymelarsan presented with higher effectiveness (100%) than aquin-treated camels (83.3%). a 3. The use of cymelarsan and CATT is recommended for disease treatment and control.