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This review does not intend to convey detailed experimental or bibliographic data. Instead, it expresses the informal authors' personal views on topics that range from basic research on antigens and experimental models for Trypanosoma cruzi infection to vaccine prospects and vaccine production. The review also includes general aspects of Chagas' disease control and international and national policies on the subject. The authors contributed equally to the paper.

Recurrent outbreaks of oral infection and isolated cases characterize the new epidemiological scenario of Chagas disease (CD) in the Brazilian Amazon. Acute Chagas disease (ACD) is common in Pará and Amazonas, Northeastern and Northwestern Brazilian Amazonia. In the present study, we describe the first molecularly characterized autochthonous case of ACD in Rondônia, Southwestern Amazonia. The patient, a 39-year-old male resident in the small city of Cujubim, presented typical ACD symptoms: fever, asthenia, myalgia, progressive dyspnea, swelling of the legs, and tiredness at minimal efforts, all compatible with ACD and indicative of cardiac involvement. A thick blood drop test revealed trypomastigote forms of Trypanosoma cruzi genotyped as TcIV. An epidemiological investigation ruled out oral infection, and support for vectorial transmission included the finding of Panstrongylus geniculatus positive for T. cruzi (TcIII and TcIV) inside the tent used by the patient when harvesting forest timber, and a circular cutaneous lesion resembling a chagoma of inoculation. Treatment with benznidazole led to blood parasite clearance as confirmed by molecular tests. Altogether, our findings fitted well into the ecological scenario where deforestation and colonization of forested areas represent an important risk factor to the adaptation of P. geniculatus to human habitats, favoring vectorial transmission of CD in the Amazonian region.

Trypanosoma rangeli and Trypanosoma cruzi are generalist trypanosomes sharing a wide range of mammalian hosts; they are transmitted by triatomine bugs, and are the only trypanosomes infecting humans in the Neotropics. Their origins, phylogenetic relationships, and emergence as human parasites have long been subjects of interest. In the present study, taxon-rich analyses (20 trypanosome species from bats and terrestrial mammals) using ssrRNA, glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH), heat shock protein-70 (HSP70) and Spliced Leader RNA sequences, and multilocus phylogenetic analyses using 11 single copy genes from 15 selected trypanosomes, provide increased resolution of relationships between species and clades, strongly supporting two main sister lineages: lineage Schizotrypanum, comprising T. cruzi and bat-restricted trypanosomes, and Tra[Tve-Tco] formed by T. rangeli, Trypanosoma vespertilionis and Trypanosoma conorhini clades. Tve comprises European T. vespertilionis and African T. vespertilionis-like of bats and bat cimicids characterised in the present study and Trypanosoma sp. Hoch reported in monkeys and herein detected in bats. Tco included the triatomine-transmitted tropicopolitan T. conorhini from rats and the African NanDoum1 trypanosome of civet (carnivore). Consistent with their very close relationships, Tra[Tve-Tco] species shared highly similar Spliced Leader RNA structures that were highly divergent from those of Schizotrypanum. In a plausible evolutionary scenario, a bat trypanosome transmitted by cimicids gave origin to the deeply rooted Tra[Tve-Tco] and Schizotrypanum lineages, and bat trypanosomes of diverse genetic backgrounds jumped to new hosts. A long and independent evolutionary history of T. rangeli more related to Old World trypanosomes from bats, rats, monkeys and civets than to Schizotrypanum spp., and the adaptation of these distantly related trypanosomes to different niches of shared mammals and vectors, is consistent with the marked differences in transmission routes, life-cycles and host-parasite interactions, resulting in T. cruzi (but not T. rangeli) being pathogenic to humans., (Copyright © 2018 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Background: Bat trypanosomes are implicated in the evolution of the T. cruzi clade, which harbours most African, European and American trypanosomes from bats and other trypanosomes from African, Australian and American terrestrial mammals, including T. cruzi and T. rangeli, the agents of the American human trypanosomiasis. The diversity of bat trypanosomes globally is still poorly understood, and the common ancestor, geographical origin, and evolution of species within the T. cruzi clade remain largely unresolved., Methods: Trypanosome sequences were obtained from cultured parasites and from museum archived liver/blood samples of bats captured from Guatemala (Central America) to the Brazilian Atlantic Coast. Phylogenies were inferred using Small Subunit (SSU) rRNA, glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH), and Spliced Leader (SL) RNA genes., Results: Here, we described Trypanosoma wauwau n. sp. from Pteronotus bats (Mormoopidae) placed in the T. cruzi clade, then supporting the bat-seeding hypothesis whereby the common ancestor of this clade likely was a bat trypanosome. T. wauwau was sister to the clade T. spp-Neobats from phyllostomid bats forming an assemblage of trypanosome species exclusively of Noctilionoidea Neotropical bats, which was sister to an Australian clade of trypanosomes from indigenous marsupials and rodents, which possibly evolved from a bat trypanosome. T. wauwau was found in 26.5% of the Pteronotus bats examined, and phylogeographical analysis evidenced the wide geographical range of this species. To date, this species was not detected in other bats, including those that were sympatric or shared shelters with Pteronotus. T. wauwau did not develop within mammalian cells, and was not infective to Balb/c mice or to triatomine vectors of T. cruzi and T. rangeli., Conclusions: Trypanosoma wauwau n. sp. was linked to Pteronotus bats. The positioning of the clade T. wauwau/T.spp-Neobats as the most basal Neotropical bat trypanosomes and closely related to an Australian lineage of trypanosomes provides additional evidence that the T. cruzi clade trypanosomes likely evolved from bats, and were dispersed in bats within and between continents from ancient to unexpectedly recent times.

Trypanosoma cruzi is a complex of phenotypically and genetically diverse isolates distributed in six discrete typing units (DTUs) designated as TcI-TcVI. Five years ago, T. cruzi isolates from Brazilian bats showing unique patterns of traditional ribosomal and spliced leader PCRs not clustering into any of the six DTUs were designated as the Tcbat genotype. In the present study, phylogenies inferred using SSU rRNA (small subunit of ribosomal rRNA), gGAPDH (glycosomal glyceraldehyde 3-phosphate dehydrogenase) and Cytb (cytochrome b) genes strongly supported Tcbat as a monophyletic lineage prevalent in Brazil, Panama and Colombia. Providing strong support for Tcbat, sequences from 37 of 47 nuclear and 12 mitochondrial genes (retrieved from a draft genome of Tcbat) and reference strains of all DTUs available in databanks corroborated Tcbat as an independent DTU. Consistent with previous studies, multilocus analysis of most nuclear genes corroborated the evolution of T. cruzi from bat trypanosomes its divergence into two main phylogenetic lineages: the basal TcII; and the lineage clustering TcIV, the clade comprising TcIII and the sister groups TcI-Tcbat. Most likely, the common ancestor of Tcbat and TcI was a bat trypanosome. However, the results of the present analysis did not support Tcbat as the ancestor of all DTUs. Despite the insights provided by reports of TcIII, TcIV and TcII in bats, including Amazonian bats harbouring TcII, further studies are necessary to understand the roles played by bats in the diversification of all DTUs. We also demonstrated that in addition to value as molecular markers for DTU assignment, Cytb, ITS rDNA and the spliced leader (SL) polymorphic sequences suggest spatially structured populations of Tcbat. Phylogenetic and phylogeographical analyses, multiple molecular markers specific to Tcbat, and the degrees of sequence divergence between Tcbat and the accepted DTUs strongly support the definitive classification of Tcbat as a new DTU., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: Bat trypanosomes have been implicated in the evolutionary history of the T. cruzi clade, which comprises species from a wide geographic and host range in South America, Africa and Europe, including bat-restricted species and the generalist agents of human American trypanosomosis T. cruzi and T. rangeli., Methods: Trypanosomes from bats (Rhinolophus landeri and Hipposideros caffer) captured in Mozambique, southeast Africa, were isolated by hemoculture. Barcoding was carried out through the V7V8 region of Small Subunit (SSU) rRNA and Fluorescent Fragment Length barcoding (FFLB). Phylogenetic inferences were based on SSU rRNA, glyceraldehyde phosphate dehydrogenase (gGAPDH) and Spliced Leader (SL) genes. Morphological characterization included light, scanning and transmission electron microscopy., Results: New trypanosomes from bats clustered together forming a clade basal to a larger assemblage called the T. cruzi clade. Barcoding, phylogenetic analyses and genetic distances based on SSU rRNA and gGAPDH supported these trypanosomes as a new species, which we named Trypanosoma livingstonei n. sp. The large and highly polymorphic SL gene repeats of this species showed a copy of the 5S ribosomal RNA into the intergenic region. Unique morphological (large and broad blood trypomastigotes compatible to species of the subgenus Megatrypanum and cultures showing highly pleomorphic epimastigotes and long and slender trypomastigotes) and ultrastructural (cytostome and reservosomes) features and growth behaviour (when co-cultivated with HeLa cells at 37°C differentiated into trypomastigotes resembling the blood forms and do not invaded the cells) complemented the description of this species., Conclusion: Phylogenetic inferences supported the hypothesis that Trypanosoma livingstonei n. sp. diverged from a common ancestral bat trypanosome that evolved exclusively in Chiroptera or switched at independent opportunities to mammals of several orders forming the clade T. cruzi, hence, providing further support for the bat seeding hypothesis to explain the origin of T. cruzi and T. rangeli.

The perspectives for a Chagas Disease vaccine 30 years ago and today are compared. Antigens and adjuvants have improved, but logistic problems remain the same. Sterilizing vaccines have not been produced and animal models for chronic Chagas have not been developed. Vector control has been successful and Chagas incidence has come to a halt. We do not have a population candidate to vaccination now in Brazil. And if we had, we would not know how to evaluate the success of vaccination in a short time period. A vaccine may not seem important at the moment. However, scientific reasons and incertitudes about the future recommend that a search for a vaccine be continued.

In this study, we provide phylogenetic and biogeographic evidence that the Trypanosoma cruzi lineages T. cruzi I (TCI) and T. cruzi IIa (TCIIa) circulate amongst non-human primates in Brazilian Amazonia, and are transmitted by Rhodnius species in overlapping arboreal transmission cycles, sporadically infecting humans. TCI presented higher prevalence rates, and no lineages other than TCI and TCIIa were found in this study in wild monkeys and Rhodnius from the Amazonian region. We characterised TCI and TCIIa from wild primates (16 TCI and five TCIIa), Rhodnius spp. (13 TCI and nine TCIIa), and humans with Chagas disease associated with oral transmission (14 TCI and five TCIIa) in Brazilian Amazonia. To our knowledge, TCIIa had not been associated with wild monkeys until now. Polymorphisms of ssrDNA, cytochrome b gene sequences and randomly amplified polymorphic DNA (RAPD) patterns clearly separated TCIIa from TCIIb-e and TCI lineages, and disclosed small intra-lineage polymorphisms amongst isolates from Amazonia. These data are important in understanding the complexity of the transmission cycles, genetic structure, and evolutionary history of T. cruzi populations circulating in Amazonia, and they contribute to both the unravelling of human infection routes and the pathological peculiarities of Chagas disease in this region.

Parasites of wild primates are important for conservation biology and human health due to their high potential to infect humans. In the Amazon region, non-human primates are commonly infected by Trypanosoma cruzi and T. rangeli, which are also infective to man and several mammals. This is the first survey of trypanosomiasis in a critically endangered species of tamarin, Saguinus bicolor (Callitrichidae), from the Brazilian Amazon Rainforest. Of the 96 free-ranging specimens of S. bicolor examined 45 (46.8%) yielded blood smears positive for trypanosomes. T. rangeli was detected in blood smears of 38 monkeys (39.6%) whereas T. cruzi was never detected. Seven animals (7.3%) presented trypanosomes of the subgenus Megatrypanum. Hemocultures detected 84 positive tamarins (87.5%). Seventy-two of 84 (85.7%) were morphologically diagnosed as T. rangeli and 3 (3.1%) as T. cruzi. Nine tamarins (9.4%) yielded mixed cultures of these two species, which after successive passages generated six cultures exclusively of T. cruzi and two of T. rangeli, with only one culture remaining mixed. Of the 72 cultures positive for T. rangeli, 62 remained as established cultures and were genotyped: 8 were assigned to phylogenetic lineage A (12.9%) and 54 to lineage B (87.1%). Ten established cultures of T. cruzi were genotyped as TCI lineage (100%). Transmission of both trypanosome species, their potential risk to this endangered species and the role of wild primates as reservoirs for trypanosomes infective to humans are discussed.

The ribosomal RNA genes of two species of Trypanosoma, Trypanosoma cruzi, the etiological agent of Chagas' disease, and Trypanosoma conorhini, a non-pathogenic rodent trypanosome, were cloned and partially characterized. The physical maps derived for their rRNA genes were similar throughout the region that encompasses the SSU-and LSU-rRNA coding sequences. However, the non-transcribed spacer DNA of both T. cruzi and T. conorhini was found to be polymorphic for several restriction enzyme sites. We show that strains of T. cruzi can be typed according to the characteristic restriction fragment length polymorphism of their NTS DNAs.

Cell extracts of culture forms of Trypanosoma cruzi are capable of hydrolysing substances belonging to 4 different groups of protease substrates: (a) substrates for trypsin-like enzymes: benzoyl-arginine-p-nitroanilide and benzoylarginine-naphtylamide; (b) substrates for aminopeptidases: leucyl, lysl and glutamyl-beta-naphtylamide; (c) a substrate fochymotrypsin-like enzymes: carbobenzoxy-L-tyrosine-p-nitorphenylester, and (d) a nonspecific substrate for a broad range of proteases: azocasein. Some physico-chemical characteristics of each enzymic reaction were studied. They were found to be distint enought to allow attributing each hydrolytic activity to a separate enzyme.

Three different methods for the radioiodination of surface proteins in trypanosomatids have been comparatively analyzed. Data interpretation in the lactoperoxidase-mediated method was impaired by autoiodination of the enzyme and its tight retention by the cells. In the enzymobeads method, iodinated enzymes were released from the beads upon solubilization of the preparation with SDS, thus mixing with iodinated proteins of the trypanosomatids. Therefore, upon separation by polyacrylamide gel electrophoresis of the cell lysates, both methods yielded autoradiographic patterns in which prominent bands did not represent labeled cell surface components. In contrast, the autoradiographic patterns obtained by use of the Iodo-Gen method were apparently free of artifacts.

Immunological cross-reactivity between Trypanosoma cruzi and insect trypanosomatids was demonstrated by immunofluorescence and confirmed by complement fixation, direct agglutination and cross-absorption experiments. As antigens, the following organisms were surveyed: Crithidia deanei, Crithidia fasciculata, Crithidia luciliae, Herpetomonas samuelpessoai, Herpetomonas megaseliae, Herpetomonas muscarum muscarum, Leptomonas seymouri and Blastocrithidia culicis. Sera from patients with Chagas' disease or sera from rabbits immunized against various trypanosomatids were used as sources of antibodies. The demonstration of cross-reactivity was followed by a survey of 500 human sera (from normal persons or Chagas' disease patients) by immunofluorescence using insect trypanosomatids (H. muscarum muscarum, C. fasciculata and L. seymouri) as antigens. With H. muscarum muscarum 98.7% coincident positive results and 100% of coincident negative results were obtained. These findings may validate the use of insect trypanosomatids as an alternative source of antigen in the serodiagnosis of Chagas' disease by indirect immunofluorescence.

Species of trypanosomatids without endosymbionts (Leptomonas seymouri, L. collosoma, L. samueli, crithidia fasciculata, C. luciliae, C. acanthocephali, Herpetomonas megaseliae, H. mariadeanei, H. samuelpessoai, H. muscarum muscarum, Trypanosoma cruzi) and species of trypanosomatids with endosymbionts (Crithidia deanei, C. oncopelti, Blastocrithidia culicis) were comparatively studied by means of electron microscopy. Artificially aposymbiotic strains derived from species with symbiont were also included in the survey. Species with symbiont were found to differ in some ultrastructural aspects from the group of species without symbiont. Paraxial rods of flagella or intraflagellar structure were found exclusively in species without symbiont. Peripheral branching of mitochondria, accompanied by absence of subpellicular microtubules in sites where the mitochondrial branches are appressed to the cell membrane, were found exclusively in species with symbiont. Networks of kinetoplast DNA fibrils were found to be larger and looser in species with symbiont. Symbiont-free strains of species with symbiont retained the same morphological characteristics of their parental species.

A simple method was developed for the characterization of different strains of Trypanosoma cruzi. T. cruzi stocks isolated from vectors or by hemoculture from patients with Chagas disease could be grouped in subpopulations having similar patterns of restriction endonuclease products of kinetoplast DNA minicircles. We designate such subpopulations by the term "schizodemes." Furthermore, it is shown that, from a given T. cruzi strain, clones with different biological properties can be isolated and identified by their restriction patterns.