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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.

Background: The subgenus Megatrypanum Hoare, 1964 of Trypanosoma Gruby, 1843 comprises trypanosomes of cervids and bovids from around the world. Here, the white-tailed deer Odocoileus virginianus (Zimmermann) and its ectoparasite, the deer ked Lipoptena mazamae Rondani, 1878 (hippoboscid fly), were surveyed for trypanosomes in Venezuela., Results: Haemoculturing unveiled 20% infected WTD, while 47% (7/15) of blood samples and 38% (11/29) of ked guts tested positive for the Megatrypanum-specific TthCATL-PCR. CATL and SSU rRNA sequences uncovered a single species of trypanosome. Phylogeny based on SSU rRNA and gGAPDH sequences tightly cluster WTD trypanosomes from Venezuela and the USA, which were strongly supported as geographical variants of the herein described Trypanosoma (Megatrypanum) trinaperronei n. sp. In our analyses, the new species was closest to Trypanosoma sp. D30 from fallow deer (Germany), both nested into TthII alongside other trypanosomes from cervids (North American elk and European fallow, red and sika deer), and bovids (cattle, antelopes and sheep). Insights into the life-cycle of T. trinaperronei n. sp. were obtained from early haemocultures of deer blood and co-culture with mammalian and insect cells showing flagellates resembling Megatrypanum trypanosomes previously reported in deer blood, and deer ked guts. For the first time, a trypanosome from a cervid was cultured and phylogenetically and morphologically (light and electron microscopy) characterised., Conclusions: In the analyses based on SSU rRNA, gGAPDH, CATL and ITS rDNA sequences, neither cervids nor bovids trypanosomes were monophyletic but intertwined within TthI and TthII major phylogenetic lineages. One host species can harbour more than one species/genotype of trypanosome, but each trypanosome species/genotype was found in a single host species or in phylogenetically closely related hosts. Molecular evidence that L. mazamae may transmit T. trinaperronei n. sp. suggests important evolutionary constraints making tight the tripartite T. trinaperronei-WTD-deer ked association. In a plausible evolutionary scenario, T. trinaperronei n. sp. entered South America with North American white-tailed deer at the Pliocene-Pleistocene boundary following the closure of the Panama Isthmus.

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.)

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.

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.

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.

A new method, the staphylococci adherence test (SAT), for the serological diagnosis of Chagas' disease is described; it is based on the affinity of staphylococcal Protein A for IgG globulins and uses epimastigotes of Trypanosoma cruzi which are fixed on to glass slides and incubated stepwise with probing sera and a Staphylococcus aureus A suspension. After Giemsa staining, epimastigote preparations which have been incubated with positive sera appear covered by cocci. Results using indirect immunofluorescence, complement fixation and SAT were in agreement in 98.37% of 860 human sera. A slightly modified SAT (SAT') may also be utilized for the diagnosis of acute Chagas' disease. The simplicity of the method may allow its adaptation for field work. Other possible uses were investigated.