Abstract. The epidemiology of malaria in 2 riverine localities in Rondônia, Brazilian western Amazônia, was assessed by a 1-year study at Portuchuelo, and a cross-sectional survey at riverine communities at Rio Machado ‫ס(‬ Ji-Paraná). Plasmodium spp. infections were diagnosed by light microscopy and by polymerase chain reaction (PCR) amplification of ribosomal DNA. PCR was 6-7 times more efficient than microscopy for detecting plasmodial infections. Both Plasmodium vivax and Plasmodium falciparum infections occurred as asymptomatic and symptomatic forms of the disease. The relation between symptomatic and asymptomatic clinical forms was roughly similar for both species of Plasmodium. Symptomless patients were monitored for 2 months. The prevalence of symptomless infections was 4-5 times higher than the symptomatic ones-respectively, 20% and 4.6% for Portuchuelo and 49.5% and 10% for JiParaná. Symptomatic malaria occurred mostly in patients in younger age groups. In contrast, there was a significant association of symptomless malaria with older age groups (medians of 26.5 and 21 years, respectively, for Portuchuelo and Ji-Paraná), whereas the age medians for symptomatic malaria were 14 and 8 years, respectively, in the 2 regions. Symptomatic malaria also was more prevalent in groups living for shorter times in Amazônia (13 and 4 years, respectively, for Portuchuelo and Ji-Paraná) as compared with symptomless malaria, which was more prevalent in groups living for longer periods in the region (medians of 25.5 and 18 years, respectively, for Portuchuelo and Ji-Paraná). The high prevalence of symptomless malaria may pose new problems for the currently adopted strategy for the control of malaria in the Amazonian region, which is essentially based on the treatment of symptomatic patients.

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We propose a taxonomic revision of the dixenous trypanosomatids currently classified as Endotrypanum and Leishmania, including parasites that do not fall within the subgenera L. (Leishmania) and L. (Viannia) related to human leishmaniasis or L. (Sauroleishmania) formed by leishmanias of lizards: L. colombiensis, L. equatorensis, L. herreri, L. hertigi, L. deanei, L. enriettii and L. martiniquensis. The comparison of these species with newly characterized isolates from sloths, porcupines and phlebotomines from central and South America unveiled new genera and subgenera supported by past (RNA PolII gene) and present (V7V8 SSU rRNA, Hsp70 and gGAPDH) phylogenetic analyses of the organisms. The genus Endotrypanum is restricted to Central and South America, comprising isolates from sloths and transmitted by phlebotomines that sporadically infect humans. This genus is the closest to the new genus Porcisia proposed to accommodate the Neotropical porcupine parasites originally described as L. hertigi and L. deanei. A new subgenus Leishmania (Mundinia) is created for the L. enriettii complex that includes L. martiniquensis. The new genus Zelonia harbours trypanosomatids from Neotropical hemipterans placed at the edge of the Leishmania-Endotrypanum-Porcisia clade. Finally, attention is drawn to the status of L. siamensis and L. australiensis as nomem nudums.

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BackgroundBat 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.MethodsTrypanosomes 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.ResultsNew 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.ConclusionPhylogenetic 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.

A simple method was developed for the characterization of different strains of Tiypanosoma 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.The hemoflagellate Trypanosoma cruzi is the etiological agent of Chagas disease, a pleomorphic clinical entity that is an important cause of morbidity and mortality in Central and South America. In some patients the infection is devastating from the very beginning with death occurring after a short acute phase; meningoencephalitis and myocarditis are prominent findings. In other cases, however, the acute phase is oligosymptomatic or even silent and may evolve without detectable sequels. Between these two extremes, in most cases the course has a variable acute phase which subsides in a few weeks, to be followed years later by digestive or cardiac symptoms or both (1).The exact causes of this clinical pleomorphism are not known, although evidence suggests that both host and parasite factors may be involved (1). In terms of the parasite, differences have been found among several strains of T. cruzi in regard to morphology, virulence, pathogenicity, and tissue tropism (1). Particularly well documented are the differences between the "polar" Y (2) and CL (3) strains. Isozyme analysis has also been used to distinguish strains of T. cruzi. Miles et al. (4-6) examined T. cruzi stocks isolated from humans and sylvatic animals in Brazil and found three distinct isozyme groups (or zymodemes). Romanha et al. (7,8) studied T. cruzi cultures obtained from humans in the city of Bambui (Minas Gerais, Brazil) and were able to group the stocks into four isozyme groups. Variations in kinetoplast DNA (kDNA) buoyant density between strains of T. cruzi and strains and species of Leishmania have been reported, and this classification method has been used by Chance (9) and Baker et al. (10).Comparison of restriction endonuclease-generated fragments of kDNA has been proposed as another method for the intrinsic classification of trypanosomes (11,12). We show here that stocks of T. cruzi isolated from patients with Chagas disease can be characterized by restriction digests of the kDNA networks. MATERIALS AND METHODSCells. With the exception of the strains Y and CL (13), all the T. cruzi stocks used in this study were obtained from patients who had positive serologic tests against Chagas disease and who were from the city of Bambui (Minas Gerais, Brazil). Stock cultures were initiated with cells-from 30 ml of blood, grown to stationary phase in liver infusion/tryptose medium (14) at 270C, and stored frozen in liquid nitrogen. Clinical histories are available for each human isolate used in this study...