Pharmacokinetic and pharmacodynamic responses in adult patients with Chagas disease treated with a new formulation of benznidazole

Pharmacological treatment of Chagas disease with benznidazole (BNZ) is effective inchildren in all stages, but it is controversial in chronically infected adults. Wereport the pharmacokinetics and pharmacodynamics in six adult patients with Chagasdisease treated with the new BNZ formulation (ABARAX^®) in doses between2.5-5.5 mg/Kg/day. All but one patient had plasmatic BNZ concentrations within theexpected range. All patients finalised treatment with nondetectableTrypanosoma cruziquantitative polymerase chain reaction, whichremained nondetectable at the six month follow-up. Our data suggests parasitologicalresponses with the new BNZ and supports the hypothesis that treatment protocols withlower BNZ doses may be effective.     

Trypanosoma cruzi: acute and long-term infection in the vertebrate host can modify the response to benznidazole

We analyzed the influence of Trypanosoma cruzi maintenance in different hosts (dog and mouse) on its susceptibility to benznidazole treatment. Five T. cruzi stocks were isolated from dogs inoculated with Be-62 or Be-78 strain (both sensitive to benznidazole) 2-10 years ago, and the benznidazole sensitivity was then determined using the mouse as experimental model. The different T. cruzi stocks obtained from long-term infected dogs showed 50-90% drug resistance right after isolation. However, maintenance of these T. cruzi stocks in mice, by successive blood passages (2.5 years), led to either a decrease or stability of the drug resistance pattern and an increase in parasite virulence. We also demonstrated the effectiveness of the induction of parasitemia reactivation by cyclophosphamide immunosuppression in the evaluation of the response to the specific drug treatment.     

Congenital transmission of Trypanosoma cruzi in central Brazil. A study of 1,211 individuals born to infected mothers

Transmission of Trypanosoma cruzi during pregnancy is estimated tooccur in less than 20% of infected mothers; however, the etiopathogenesis is notcompletely understood. The Centre for Studies on Chagas Disease provides confirmationof T. cruzi infection for individuals living in central Brazil.In this retrospective hospital-based study, all requests for diagnosis of T.cruzi infection in individuals less than 21 years old from 1994-2014 weresearched. We end with 1,211 individuals and their respective infected mothers.Congenital transmission of infection was confirmed in 24 individuals (2%) in centralBrazil, an area where the main T. cruzi lineage circulating inhumans is TcII. This low prevalence of congenital Chagas disease is discussed inrelation to recent findings in the south region of Brazil, where TcV is the mainlineage and congenital transmission has a higher prevalence (approximately 5%),similar to frequencies reported in Argentina, Paraguay and Bolivia. This is the firstreport to show geographical differences in the rates of congenital transmissionof T. cruzi and the relationship between the prevalence ofcongenital transmission and the type of Tc prevalent in each region.     

Retrospective distribution of Trypanosoma cruzi I genotypes in Colombia

Trypanosoma cruzi is the aetiological agent of Chagas disease, whichaffects approximately eight million people in the Americas. This parasite exhibitsgenetic variability, with at least six discrete typing units broadly distributed inthe American continent. T. cruzi I (TcI) shows remarkable geneticdiversity; a genotype linked to human infections and a domestic cycle of transmissionhave recently been identified, hence, this strain was named TcIDom. The aim of thiswork was to describe the spatiotemporal distribution of TcI subpopulations acrosshumans, insect vectors and mammalian reservoirs in Colombia by means of moleculartyping targeting the spliced leader intergenic region of mini-exon gene. We analysed101 TcI isolates and observed a distribution of sylvatic TcI in 70% and TcIDom in30%. In humans, the ratio was sylvatic TcI in 60% and TcIDom in 40%. In mammalreservoirs, the distribution corresponded to sylvatic TcI in 96% and TcIDom in 4%.Among insect vectors, sylvatic TcI was observed in 48% and TcIDom in 52%. Inconclusion, the circulation of TcIDom is emerging in Colombia and this genotype isstill adapting to the domestic cycle of transmission. The epidemiological andclinical implications of these findings are discussed herein.     

Trypanosoma cruzi: Induction of benznidazole resistance in vivo and its modulation by in vitro culturing and mice infection

Through a continuous in vivo drug pressure protocol, using mice as experimental model, we induced benznidazole resistance in Trypanosoma cruzi stocks. Full resistance was obtained for four out of five T. cruzi stocks analyzed. However, the number of benznidazole doses (40–180), as well as the time (4–18 months) necessary to induce resistance varied among the different T. cruzi stocks. The resistance phenotype remained stable after T. cruzi stocks has been maintained by 12 passages in mice (six months) and in acellular culture for the same time. However, the maintenance of resistant parasite for 12 months in acellular culture induces a reduction in its level of benznidazole resistance, while no alteration was detected in parasite maintained for the same time in mice. The data showed the stability of the resistance acquired by drug pressure, but suggest the possibility of reversible changes in the resistance levels after maintenance for long time in acellular culture.     

The main sceneries of Chagas disease transmission. The vectors,blood and oral transmissions - A comprehensive review

This review deals with transmission of Trypanosoma cruzi by the most important domestic vectors, blood transfusion and oral intake. Among the vectors, Triatoma infestans, Panstrongylus megistus, Rhodnius prolixus, Triatoma dimidiata, Triatoma brasiliensis, Triatoma pseudomaculata, Triatoma sordida, Triatoma maculata, Panstrongylus geniculatus, Rhodnius ecuadoriensis and Rhodnius pallescens can be highlighted. Transmission of Chagas infection, which has been brought under control in some countries in South and Central America, remains a great challenge, particularly considering that many endemic countries do not have control over blood donors. Even more concerning is the case of non-endemic countries that receive thousands of migrants from endemic areas that carry Chagas disease, such as the United States of America, in North America, Spain, in Europe, Japan, in Asia, and Australia, in Oceania. In the Brazilian Amazon Region, since Shaw et al. (1969) described the first acute cases of the disease caused by oral transmission, hundreds of acute cases of the disease due to oral transmission have been described in that region, which is today considered to be endemic for oral transmission. Several other outbreaks of acute Chagas disease by oral transmission have been described in different states of Brazil and in other South American countries.     

The in vitro activity of fatty diamines and amino alcohols against mixed amastigote and trypomastigote Trypanosoma cruzi forms

Four diamines and three amino alcohols derived from 1-decanol, 1-dodecanol and 1,2-dodecanediol were evaluated in an in vitro assay against a mixture of trypomastigote and intracellular amastigote forms of Trypanosoma cruzi. Two of these compounds (6 and 7) showed better activity against both proliferative stages of T. cruzi than the positive control benznidazole, three were of similar potency (1, 2 and 5) and two were less active (3 and 4).     

Immunological response to re-infections with clones of the Colombian strain of Trypanosoma cruzi with different degrees of virulence: influence on pathological features during chronic infection in mice

Re-infections with Trypanosoma cruzi are an aggravating factor forChagas disease morbidity. The Colombian strain of T. cruzirepresents multiclonal populations formed by clonally propagating organisms withdifferent tropisms and degrees of virulence. In the present study, the influence ofsuccessive inoculations with clones of the Colombian strain, exhibiting differentdegrees of virulence, on chronic myocarditis and the humoral and cellular immuneresponses (Col-C1 high virulence, Col-C8 medium virulence and Col-C5 low virulence)were demonstrated. Mice from three groups with a single infection were evaluatedduring the acute (14th-30th day) and chronic phases for 175 days. Animmunofluorescence assay, ELISA and delayed type hypersensitivity (DTH) cutaneoustest were also performed. Mice with a triple infection were studied on the115th-175th days following first inoculation. The levels of IgM and IgG2a were higherin the animals with a triple infection. DTH showed a higher intensity in theinflammatory infiltrate based on the morphometric analysis during a 48 h period ofthe triple infection and at 24 h with a single infection. The histopathology of theheart demonstrated significant exacerbation of cardiac inflammatory lesions confirmedby the morphometric test. The humoral responses indicate a reaction to the tripleinfection, even with clones of the same strain.     

New scenarios of Trypanosoma cruzi transmission in the Orinoco region of Colombia

Rhodnius prolixus, a blood-sucking triatomine with domiciliary anthropophilic habits, is the main vector of Chagas disease. The current paradigm of Trypanosoma cruzi transmission in Columbia includes a sylvatic and domiciliary cycle co-existing with domestic and sylvatic populations of reservoirs. The aim of this study is to evaluate the population densities and relative abundance of triatomines and mammals that may be involved in the sylvatic cycle of Chagas disease to clarify the epidemiological scenario in an endemic area in the province of Casanare. Insect vectors on Attalea butyracea palms were captured using both manual searches and bait traps. The capture of mammals was performed using Sherman and Tomahawk traps. We report an infestation index of 88.5% in 148 palms and an index of T. cruzi natural infection of 60.2% in 269 dissected insects and 11.9% in 160 captured mammals. High population densities of triatomines were observed in the sylvatic environment and there was a high relative abundance of reservoirs in the area, suggesting a stable enzootic cycle. We found no evidence of insect domiciliation. Taken together, these observations suggest that eco-epidemiological factors shape the transmission dynamics of T. cruzi, creating diverse scenarios of disease transmission.     

Dynamic flux of microvesicles modulate parasite–host cell interaction of Trypanosoma cruzi in eukaryotic cells

Extracellular vesicles released from pathogens may alter host cell functions. We previously demonstrated the involvement of host cell‐derived microvesicles (MVs) during early interaction between Trypanosoma cruzi metacyclic trypomastigote (META) stage and THP‐1 cells. Here, we aim to understand the contribution of different parasite stages and their extracellular vesicles in the interaction with host cells. First, we observed that infective host cell‐derived trypomastigote (tissue culture‐derived trypomastigote [TCT]), META, and noninfective epimastigote (EPI) stages were able to induce different levels of MV release from THP‐1 cells; however, only META and TCT could increase host cell invasion. Fluorescence resonance energy transfer microscopy revealed that THP‐1‐derived MVs can fuse with parasite‐derived MVs. Furthermore, MVs derived from the TCT–THP‐1 interaction showed a higher fusogenic capacity than those from META– or EPI–THP‐1 interaction. However, a higher presence of proteins from META (25%) than TCT (12%) or EPI (5%) was observed in MVs from parasite–THP‐1 interaction, as determined by proteomics. Finally, sera from patients with chronic Chagas disease at the indeterminate or cardiac phase differentially recognized antigens in THP‐1‐derived MVs resulting only from interaction with infective stages. The understanding of intracellular trafficking and the effect of MVs modulating the immune system may provide important clues about Chagas disease pathophysiology.     

Mapping of B cell epitopes in an immunodominant antigen of Trypanosoma cruzi using fusions to the Escherichia coli LamB protein

The JL8 protein antigen from Trypanosoma cruzi, a dominant immunogen in man, has been characterized as containing tandem amino acid repeats. Here, we describe the use of the LamB protein of Escherichia coli as a carrier of JL8 derived sequences in order to map the immunodominant B cell epitopes in this antigen. Five different sequences of JL8 were inserted in the LamB protein and the JL8-LamB fusion proteins were tested by ELISA with human chronic chagasic sera. The fusion carrying the sequence AEKQKAAEATKVAE was recognized by most sera. This protein was also capable of inhibiting the binding of human chagasic antibodies to GST-JL8 in competitive ELISA suggesting that it contains an immunodominant B cell epitope of JL8.     

Role of cyclooxygenase-2 in Trypanosoma cruzi survival in the early stages of parasite host-cell interaction

Chagas disease, caused by the intracellular protozoan Trypanosoma cruzi, is a serious health problem in Latin America. During this parasitic infection, the heart is one of the major organs affected. The pathogenesis of tissue remodelling, particularly regarding cardiomyocyte behaviour after parasite infection and the molecular mechanisms that occur immediately following parasite entry into host cells are not yet completely understood. When cells are infected with T. cruzi, they develop an inflammatory response, in which cyclooxygenase-2 (COX-2) catalyses rate-limiting steps in the arachidonic acid pathway. However, how the parasite interaction modulates COX-2 activity is poorly understood. In this study, the H9c2 cell line was used as our model and we investigated cellular and biochemical aspects during the initial 48 h of parasitic infection. Oscillatory activity of COX-2 was observed, which correlated with the control of the pro-inflammatory environment in infected cells. Interestingly, subcellular trafficking was also verified, correlated with the control of Cox-2 mRNA or the activated COX-2 protein in cells, which is directly connected with the assemble of stress granules structures. Our collective findings suggest that in the very early stage of the T. cruzi-host cell interaction, the parasite is able to modulate the cellular metabolism in order to survives.     

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.     

A novel maize microRNA negatively regulates resistance to Fusarium verticillioides

Although microRNAs (miRNAs) regulate the defence response against multiple pathogenic fungi in diverse plant species, few efforts have been devoted to deciphering the involvement of miRNA in resistance to Fusarium verticillioides, a major pathogenic fungus affecting maize production. In this study, we discovered a novel F. verticillioides‐responsive miRNA designated zma‐unmiR4 in maize kernels. The expression of zma‐unmiR4 was significantly repressed in the resistant maize line but induced in the susceptible lines upon exposure to F. verticillioides exposure, whereas its target gene ZmGA2ox4 exhibited the opposite pattern of expression. Heterologous overexpression of zma‐unmiR4 in Arabidopsis resulted in enhanced growth and compromised resistance to F. verticillioides. By contrast, transgenic plants overexpressing ZmGA2ox4 or the homologue AtGA2ox7 showed impaired growth and enhanced resistance to F. verticillioides. Moreover, zma‐unmiR4‐mediated suppression of AtGA2ox7 disturbed the accumulation of bioactive gibberellin (GA) in transgenic plants and perturbed the expression of a set of defence‐related genes in response to F. verticillioides. Exogenous application of GA or a GA biosynthesis inhibitor modulated F. verticillioides resistance in different plants. Taken together, our results suggest that the zma‐unmiR4–ZmGA2ox4 module might act as a major player in balancing growth and resistance to F. verticillioides in maize.     

Trypanosoma cruzi: role of the immune response in the natural resistance of inbred strains of mice.

Nine inbred strains of mice were challenged with 10⁴ or 10⁵ trypomastigotes of the Brazil strain of Trypanosoma cruzi. A spectrum of resistance was evident ranging from highly susceptible strains, e.g., C3H, which developed high parasitemias and died within 3 to 4 weeks, to resistant strains, e.g., C57BL/10, which developed low parasitemias and survived. Impairment of the immune system in resistant C57BL/10 mice by X-irradiation, splenectomy, or treatment with silica led to high, often fatal parasitemias. Athymic nude mice, in particular, attained exceptionally high parasitemias before dying. The immune response appears to be necessary for survival and to play a role in the natural resistance of some mouse strains by effectively eliminating parasites and minimizing parasitemia. Using congenic strains of mice, it was shown that the principal genetic determinant of resistance is not associated with their H-2 haplotype.