Aptamer-Based Detection of Disease Biomarkers in Mouse Models for Chagas Drug Discovery

Drug discovery initiatives, aimed at Chagas treatment, have been hampered by the lack of standardized drug screening protocols and the absence of simple pre-clinical assays to evaluate treatment efficacy in animal models. In this study, we used a simple Enzyme Linked Aptamer (ELA) assay to detect T. cruzi biomarker in blood and validate murine drug discovery models of Chagas disease. In two mice models, Apt-29 ELA assay demonstrated that biomarker levels were significantly higher in the infected group compared to the control group, and upon Benznidazole treatment, their levels reduced. However, biomarker levels in the infected treated group did not reduce to those seen in the non-infected treated group, with 100% of the mice above the assay cutoff, suggesting that parasitemia was reduced but cure was not achieved. The ELA assay was capable of detecting circulating biomarkers in mice infected with various strains of T. cruzi parasites. Our results showed that the ELA assay could detect residual parasitemia in treated mice by providing an overall picture of the infection in the host. They suggest that the ELA assay can be used in drug discovery applications to assess treatment efficacy in-vivo.     

Coronary Microvascular Disease in Chronic Chagas Cardiomyopathy Including an Overview on History,Pathology, and Other Proposed Pathogenic Mechanisms

This review focuses on the short and bewildered history of Brazilian scientist Carlos Chagas''s discovery and subsequent developments, the anatomopathological features of chronic Chagas cardiomyopathy (CCC), an overview on the controversies surrounding theories concerning its pathogenesis, and studies that support the microvascular hypothesis to further explain the pathological features and clinical course of CCC. It is our belief that knowledge of this particular and remarkable cardiomyopathy will shed light not only on the microvascular involvement of its pathogenesis, but also on the pathogenetic processes of other cardiomyopathies, which will hopefully provide a better understanding of the various changes that may lead to an end-stage heart disease with similar features. This review is written to celebrate the 100th anniversary of the discovery of Chagas disease.     

Optimization and evaluation of the ARCHITECT Chagas assay and in-house ELISA for Chagas disease in clinical settings in Japan

Approximately 250,000 immigrants from Latin America live in Japan and it is estimated that 1500–3000 of them are potentially infected with Trypanosoma cruzi, the cause of Chagas disease. Therefore, the establishment of a standardized diagnostic method for Chagas disease in Japan is urgently needed. In this study, we optimized and evaluated the ARCHITECT Chagas assay and in-house ELISA for Chagas disease in clinical settings. In particular, we evaluated the performance of ARCHITECT Chagas as well as ELISA with whole-cell lysates and three recombinant proteins (TcF, TcBCDE, and CP1 + CP3) using 93 Chagas disease-positive serum samples and 108 Chagas disease-positive samples. The sensitivities of ARCHITECT Chagas, whole-cell lysate, TcF, TcBCDE, and CP1 + CP3 ELISA were respectively 100%, 100%, 98.9%, 98.9%, and 89.2% and the corresponding specificities were 100%, 99.1%, 99.1%, 100%, and 99.1%. False-positive results were obtained for whole-cell lysate, TcF, and CP1 ± CP3 ELISA. This is the first evidence that OD cut-off values optimized for in-house ELISA are similar in terms of sensitivity and specificity to those of the ARCHITECT Chagas test, supporting the use of these in-house assays as diagnostic tests for Chagas disease in the clinical setting in Japan.     

Discovery and characterization of an antibody, anti-egressin, that is able to inhibit Trypanosoma cruzi egress in vitro

Trypanosoma cruzi, a protozoan parasite, is the etiologic agent of Chagas disease. The disease is characterized by acute and chronic phases, with high and low parasitemia, respectively. A strong immune activation is necessary for the host to enter the chronic phase; however, immune mechanisms participating in the reduction of parasites between the acute and chronic phases of the disease have been very difficult to elucidate. We report here the discovery of anti-egressin, an antibody present in serum from chronically infected BALB/c mice that is able to inhibit parasite egress from infected BALB/c fibroblast cultures in vitro. The antibody is very concentrated in serum from these mice; chronic serum may be diluted 1:20 while still maintaining functional activity. Isotype analysis of anti-egressin has suggested it to be IgG2a. Further analysis revealed that anti-egressin was specific for a component expressed on the surface of infected host cells. The specificity of anti-egressin toward the extracellular portion of infected host cells was demonstrated both by using a quantitative assay measuring released trypomastigotes and through immunocytochemical staining. The novel role of anti-egressin in the inhibition of parasite egress from infected host cells has not been described in the literature to date. We believe that anti-egressin plays an important role in achieving the low parasitemia characteristic of chronic Chagas disease.     

Ecoepidemiology,short history and control of Chagas disease in the endemic countries and the new challenge for non-endemic countries

Chagas disease is maintained in nature through the interchange of three cycles: the wild, peridomestic and domestic cycles. The wild cycle, which is enzootic, has existed for millions of years maintained between triatomines and wild mammals. Human infection was only detected in mummies from 4,000-9,000 years ago, before the discovery of the disease by Carlos Chagas in 1909. With the beginning of deforestation in the Americas, two-three centuries ago for the expansion of agriculture and livestock rearing, wild mammals, which had been the food source for triatomines, were removed and new food sources started to appear in peridomestic areas: chicken coops, corrals and pigsties. Some accidental human cases could also have occurred prior to the triatomines in peridomestic areas. Thus, triatomines progressively penetrated households and formed the domestic cycle of Chagas disease. A new epidemiological, economic and social problem has been created through the globalisation of Chagas disease, due to legal and illegal migration of individuals infected by Trypanosoma cruzi or presenting Chagas disease in its varied clinical forms, from endemic countries in Latin America to non-endemic countries in North America, Europe, Asia and Oceania, particularly to the United States of America and Spain. The main objective of the present paper was to present a general view of the interchanges between the wild, peridomestic and domestic cycles of the disease, the development of T. cruzi among triatomine, their domiciliation and control initiatives, the characteristics of the disease in countries in the Americas and the problem of migration to non-endemic countries.     

Global Metabolomic Profiling of Acute Myocarditis Caused by Trypanosoma cruzi Infection

Chagas disease is caused by Trypanosoma cruzi infection, being cardiomyopathy the more frequent manifestation. New chemotherapeutic drugs are needed but there are no good biomarkers for monitoring treatment efficacy. There is growing evidence linking immune response and metabolism in inflammatory processes and specifically in Chagas disease. Thus, some metabolites are able to enhance and/or inhibit the immune response. Metabolite levels found in the host during an ongoing infection could provide valuable information on the pathogenesis and/or identify deregulated metabolic pathway that can be potential candidates for treatment and being potential specific biomarkers of the disease. To gain more insight into those aspects in Chagas disease, we performed an unprecedented metabolomic analysis in heart and plasma of mice infected with T. cruzi. Many metabolic pathways were profoundly affected by T. cruzi infection, such as glucose uptake, sorbitol pathway, fatty acid and phospholipid synthesis that were increased in heart tissue but decreased in plasma. Tricarboxylic acid cycle was decreased in heart tissue and plasma whereas reactive oxygen species production and uric acid formation were also deeply increased in infected hearts suggesting a stressful condition in the heart. While specific metabolites allantoin, kynurenine and p-cresol sulfate, resulting from nucleotide, tryptophan and phenylalanine/tyrosine metabolism, respectively, were increased in heart tissue and also in plasma. These results provide new valuable information on the pathogenesis of acute Chagas disease, unravel several new metabolic pathways susceptible of clinical management and identify metabolites useful as potential specific biomarkers for monitoring treatment and clinical severity in patients.     

Prophet in his own country: Carlos Chagas and the Nobel Prize

In 1909, Carlos Chagas (1878-1934) discovered a new protozoon, Trypanosoma cruzi, and the (previously unknown) disease that it causes. Within a few months, virtually single-handed, he described the pathogen, its vector, and the clinical features of American trypanosomiasis (Chagas disease), a feat unique in medical history. He headed the Oswaldo Cruz Institute after the death of its founder (1917) until his own death; and from 1920 until 1926 he also directed the Brazilian Department of Public Health. His discovery brought him worldwide acclaim, but at home antagonism against Chagas, muted for years, finally flared up in a campaign that was acted out in the 1921-22 plenary sessions of the National Academy of Medicine. Chagas's name was repeatedly proposed for the Nobel Prize but he never received it; this hostile campaign may have been instrumental in costing him the award.     

Severity of chronic Chagas disease is associated with cytokine/antioxidant imbalance in chronically infected individuals

Understanding the pathogenic mechanisms in chronic Chagas disease, a major cause of morbidity and mortality in Latin America, is essential for the design of rational therapeutic strategies. In this paper we show that the development of Chagas disease is a consequence of a long-term and complex relationship between parasite persistence and maladapted homeostatic mechanisms in the host which leads to pathologic changes. We performed a retrospective study on 50 patients with chronic Chagas disease and 50 healthy control individuals. The specific immune response was detected by ELISA and IHA tests using autochthonous antigens, inflammatory process with the cytokine tumour necrosis factor (TNF)-alpha and nitric oxide (NO), and antioxidant protection with glutathione peroxidase and superoxide dismutase (SOD) levels. We developed generalised linear modelling procedures to assess simultaneously which explanatory variables and/or their interactions better explained disease severity in patients. Our results show the existence of a strong relationship between anti-Trypanosoma cruzi levels and chronic Chagas disease (P<0.0001). Taken together, the statistical data indicate both cumulative and complementary effects, where the increase in TNF-alpha (P=0.004) and NO (P=0.005) levels correlated with a reduction in glutathione peroxidase (P=0.0001) and SOD (P=0.01) levels drives the disease pathology in chronically infected patients. Our findings may have important implications for understanding host susceptibility to develop severe chronic infectious disease. In addition we show putative targets for the design of new therapeutic strategies to prevent disease progression, considering both specific treatment against the aetiological agent and modulation of the different immunopathological reactions in chronically infected individuals with chronic Chagas disease.     

Ruthenium Complex with Benznidazole and Nitric Oxide as a New Candidate for the Treatment of Chagas Disease

Background

Chagas disease remains a serious medical and social problem in Latin America and is an emerging concern in nonendemic countries as a result of population movement, transfusion of infected blood or organs and congenital transmission. The current treatment of infected patients is unsatisfactory due to strain-specific drug resistance and the side effects of the current medications. For this reason, the discovery of safer and more effective chemotherapy is mandatory for the successful treatment and future eradication of Chagas disease.

Methods and Findings

We investigated the effect of a ruthenium complex with benznidazole and nitric oxide (RuBzNO₂) against Trypanosoma cruzi both in vitro and in vivo. Our results demonstrated that RuBzNO₂ was more effective than the same concentrations of benznidazole (Bz) in eliminating both the extracellular trypomastigote and the intracellular amastigote forms of the parasite, with no cytotoxic effect in mouse cells. In vivo treatment with the compound improved the survival of infected mice, inhibiting heart damage more efficiently than Bz alone. Accordingly, tissue inflammation and parasitism was significantly diminished after treatment with RuBzNO₂ in a more effective manner than that with the same concentrations of Bz.

Conclusions

The complexation of Bz with ruthenium and nitric oxide (RuBzNO₂) increases its effectiveness against T. cruzi and enables treatment with lower concentrations of the compound, which may reduce the side effects of Bz. Our findings provide a new potential candidate for the treatment of Chagas disease.     

Integrated control of Chagas disease for its elimination as public health problem - A Review

Chagas disease or American trypanosomiasis is, together with geohelminths, the neglected disease that causes more loss of years of healthy life due to disability in Latin America. Chagas disease, as determined by the factors and determinants, shows that different contexts require different actions, preventing new cases or reducing the burden of disease. Control strategies must combine two general courses of action including prevention of transmission to prevent the occurrence of new cases (these measures are cost effective), as well as opportune diagnosis and treatment of infected individuals in order to prevent the clinical evolution of the disease and to allow them to recuperate their health. All actions should be implemented as fully as possible and with an integrated way, to maximise the impact. Chagas disease cannot be eradicated due because of the demonstrated existence of infected wild triatomines in permanent contact with domestic cycles and it contributes to the occurrence of at least few new cases. However, it is possible to interrupt the transmission of Trypanosoma cruzi in a large territory and to eliminate Chagas disease as a public health problem with a dramatic reduction of burden of the disease.     

High Throughput Screening for Anti–Trypanosoma cruzi Drug Discovery

The discovery of new therapeutic options against Trypanosoma cruzi, the causative agent of Chagas disease, stands as a fundamental need. Currently, there are only two drugs available to treat this neglected disease, which represents a major public health problem in Latin America. Both available therapies, benznidazole and nifurtimox, have significant toxic side effects and their efficacy against the life-threatening symptomatic chronic stage of the disease is variable. Thus, there is an urgent need for new, improved anti–T. cruzi drugs. With the objective to reliably accelerate the drug discovery process against Chagas disease, several advances have been made in the last few years. Availability of engineered reporter gene expressing parasites triggered the development of phenotypic in vitro assays suitable for high throughput screening (HTS) as well as the establishment of new in vivo protocols that allow faster experimental outcomes. Recently, automated high content microscopy approaches have also been used to identify new parasitic inhibitors. These in vitro and in vivo early drug discovery approaches, which hopefully will contribute to bring better anti–T. cruzi drug entities in the near future, are reviewed here.     

The clinical immunology of human Chagas disease

Human infection with the protozoan parasite Trypanosoma cruzi leads to Chagas disease, which affects approximately 17 million people in Latin America. A significant percentage of the infected population will develop clinical symptoms or present changes in laboratory and/or image evaluation. The existence of a large spectrum of clinical manifestations--with patients ranging from asymptomatic to severe cardiac involvement--emphasizes the need to use standardized and well-defined clinical criteria among different research groups. In this article, we carry out a systematic review of the immunology in human Chagas disease, discussing recent findings in the context of a clinical perspective.     

Update on oral Chagas disease outbreaks in Venezuela: epidemiological, clinical and diagnostic approaches

Orally transmitted Chagas disease has become a matter of concern due to outbreaks reported in four Latin American countries. Although several mechanisms for orally transmitted Chagas disease transmission have been proposed, food and beverages contaminated with whole infected triatomines or their faeces, which contain metacyclic trypomastigotes of Trypanosoma cruzi, seems to be the primary vehicle. In 2007, the first recognised outbreak of orally transmitted Chagas disease occurred in Venezuela and largest recorded outbreak at that time. Since then, 10 outbreaks (four in Caracas) with 249 cases (73.5% children) and 4% mortality have occurred. The absence of contact with the vector and of traditional cutaneous and Romana’s signs, together with a florid spectrum of clinical manifestations during the acute phase, confuse the diagnosis of orally transmitted Chagas disease with other infectious diseases. The simultaneous detection of IgG and IgM by ELISA and the search for parasites in all individuals at risk have been valuable diagnostic tools for detecting acute cases. Follow-up studies regarding the microepidemics primarily affecting children has resulted in 70% infection persistence six years after anti-parasitic treatment. Panstrongylus geniculatus has been the incriminating vector in most cases. As a food-borne disease, this entity requires epidemiological, clinical, diagnostic and therapeutic approaches that differ from those approaches used for traditional direct or cutaneous vector transmission.     

Chagas cardiomyopathy manifestations and Trypanosoma cruzi genotypes circulating in chronic Chagasic patients

Chagas disease caused by Trypanosoma cruzi is a complex disease that is endemic and an important problem in public health in Latin America. The T. cruzi parasite is classified into six discrete taxonomic units (DTUs) based on the recently proposed nomenclature (TcI, TcII, TcIII, TcIV, TcV and TcVI). The discovery of genetic variability within TcI showed the presence of five genotypes (Ia, Ib, Ic, Id and Ie) related to the transmission cycle of Chagas disease. In Colombia, TcI is more prevalent but TcII has also been reported, as has mixed infection by both TcI and TcII in the same Chagasic patient. The objectives of this study were to determine the T. cruzi DTUs that are circulating in Colombian chronic Chagasic patients and to obtain more information about the molecular epidemiology of Chagas disease in Colombia. We also assessed the presence of electrocardiographic, radiologic and echocardiographic abnormalities with the purpose of correlating T. cruzi genetic variability and cardiac disease. Molecular characterization was performed in Colombian adult chronic Chagasic patients based on the intergenic region of the mini-exon gene, the 24Sα and 18S regions of rDNA and the variable region of satellite DNA, whereby the presence of T.cruzi I, II, III and IV was detected. In our population, mixed infections also occurred, with TcI-TcII, TcI-TcIII and TcI-TcIV, as well as the existence of the TcI genotypes showing the presence of genotypes Ia and Id. Patients infected with TcI demonstrated a higher prevalence of cardiac alterations than those infected with TcII. These results corroborate the predominance of TcI in Colombia and show the first report of TcIII and TcIV in Colombian Chagasic patients. Findings also indicate that Chagas cardiomyopathy manifestations are more correlated with TcI than with TcII in Colombia.     

High-content imaging for automated determination of host-cell infection rate by the intracellular parasite Trypanosoma cruzi

Chagas disease affects 8–11 million people, mostly in Latin America. Sequelae include cardiac, peripheral nervous and/or gastrointestinal disorders, thus placing a large economic and social burden on endemic countries. The pathogenesis and the evolutive pattern of the disease are not fully clarified. Moreover, available drugs are partially effective and toxic, and there is no vaccine. Therefore, there is an urgent need to speed up basic and translational research in the field. Here, we applied automated high-content imaging to generate multiparametric data on a cell-by-cell basis to precisely and quickly determine several parameters associated with in vitro infection of host cell by Trypanosoma cruzi, the causative agent of Chagas disease. Automated and manual quantifications were used to determine the percentage of T. cruzi-infected cells in a 96-well microplate format and the data generated was statistically evaluated. Most importantly, this automated approach can be widely applied for discovery of potential drugs as well as molecular pathway elucidation not only in T. cruzi but also in other human intracellular pathogens.     

The challenge of Chagas’ disease: Has the human pathogen,Trypanosoma cruzi,learned how to modulate signaling events to subvert host cells?

Chagas’ disease, caused by Trypanosoma cruzi, is an urgent and highly prevalent danger that is endemic to Latin America, and which the research community continues to ignore. Each year, Chagas’ disease kills more people in Latin America compared to any other parasite-borne disease, including malaria. In addition, between 15 and 18 million people worldwide are afflicted with this potentially lethal disease. Despite these devastating numbers, less than 0.5% of worldwide research and development for neglected diseases was aimed at Chagas’ disease. The aim of this review is to draw the attention of biotechnologists to the intriguing parasite that causes Chagas’ disease, which is T. cruzi. Additionally, we would also like to convince the community that basic science research can have a profound impact on the diagnosis and treatment of Chagas’ disease. In this review, we introduce distinct features of T. cruzi such as its complex life cycle (e.g. the potentially infective extracellular amastigote form), its genome and genomics, as well as proteomic analysis of this parasite. Notably, the PIK pathway has been widely acknowledged as an excellent target for drug discovery to combat this pathogen. Furthermore we also describe how the identification and characterization of PIK genes can aid in neutralizing Trypanosoma infections.     

Acute Chagas Disease: New Global Challenges for an Old Neglected Disease

Chagas disease is caused by infection with the protozoan Trypanosoma cruzi, and although over 100 years have passed since the discovery of Chagas disease, it still presents an increasing problem for global public health. A plethora of information concerning the chronic phase of human Chagas disease, particularly the severe cardiac form, is available in the literature. However, information concerning events during the acute phase of the disease is scarce. In this review, we will discuss (1) the current status of acute Chagas disease cases globally, (2) the immunological findings related to the acute phase and their possible influence in disease outcome, and (3) reactivation of Chagas disease in immunocompromised individuals, a key point for transplantation and HIV infection management.     

Opportunity Cost for Early Treatment of Chagas Disease in Mexico

Background

Given current neglect for Chagas disease in public health programs in Mexico, future healthcare and economic development policies will need a more robust model to analyze costs and impacts of timely clinical attention of infected populations.

Methodology/Principal Findings

A Markov decision model was constructed to simulate the natural history of a Chagas disease cohort in Mexico and to project the associated short and long-term clinical outcomes and corresponding costs. The lifetime cost for a timely diagnosed and treated Chagas disease patient is US$ 10,160, while the cost for an undiagnosed individual is US$ 11,877. The cost of a diagnosed and treated case increases 24-fold from early acute to indeterminate stage. The major cost component for lifetime cost was working days lost, between 44% and 75%, depending on the program scenario for timely diagnosis and treatment.

Conclusions/Significance

In the long term, it is cheaper to diagnose and treat chagasic patients early, instead of doing nothing. This finding by itself argues for the need to shift current policy, in order to prioritize and attend this neglected disease for the benefit of social and economic development, which implies including treatment drugs in the national formularies. Present results are even more relevant, if one considers that timely diagnosis and treatment can arrest clinical progression and enhance a chronic patient''s quality of life.     

Urban Chagas disease in children and women in primary care centres in Buenos Aires,Argentina

The primary objective of this study was to estimate the prevalence of this disease in women of childbearing age and children treated at health centres in underserviced areas of the city of Buenos Aires. Demographic and Chagas disease status data were collected. Samples for Chagas disease serology were obtained on filter paper and the reactive results were confirmed with conventional samples. A total of 1,786 subjects were screened and 73 positive screening results were obtained: 17 were from children and 56 were from women. The Trypanosoma cruzi infection risk was greater in those individuals who had relatives with Chagas disease, who remember seeing kissing bugs, who were of Bolivian nationality or were born in the Argentine province of Santiago del Estero. The overall prevalence of Chagas disease was 4.08%. Due to migration, Chagas disease is currently predominantly urban. The observed prevalence requires health programme activities that are aimed at urban children and their mothers. Most children were infected congenitally, which reinforces the need for Chagas disease screening of all pregnant women and their babies in Argentina. The active search for new cases is important because the appropriate treatment in children has a high cure rate.