BackgroundChagas disease is an emergent tropical disease in the Brazilian Amazon Region, with an increasing number of cases in recent decades. In this region, the sylvatic cycle of Trypanosoma cruzi transmission, which constitutes a reservoir of parasites that might be associated with specific molecular, epidemiological and clinical traits, has been little explored. The objective of this work is to genetically characterize stocks of T. cruzi from human cases, triatomines and reservoir mammals in the State of Amazonas, in the Western Brazilian Amazon.Methodology/Principal FindingsWe analyzed 96 T. cruzi samples from four municipalities in distant locations of the State of Amazonas. Molecular characterization of isolated parasites from cultures in LIT medium or directly from vectors or whole human blood was performed by PCR of the non-transcribed spacer of the mini-exon and of the 24 S alfa ribosomal RNA gene, RFLP and sequencing of the mitochondrial cytochrome c oxidase subunit II (COII) gene, and by sequencing of the glucose-phosphate isomerase gene. The T. cruzi parasites from two outbreaks of acute disease were all typed as TcIV. One of the outbreaks was triggered by several haplotypes of the same DTU. TcIV also occurred in isolated cases and in Rhodnius robustus. Incongruence between mitochondrial and nuclear phylogenies is likely to be indicative of historical genetic exchange events resulting in mitochondrial introgression between TcIII and TcIV DTUs from Western Brazilian Amazon. TcI predominated among triatomines and was the unique DTU infecting marsupials.Conclusion/SignificanceDTU TcIV, rarely associated with human Chagas disease in other areas of the Amazon basin, is the major strain responsible for the human infections in the Western Brazilian Amazon, occurring in outbreaks as single or mixed infections by different haplotypes.

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BackgroundThe parasites Trypanosoma rangeli and Trypanosoma cruzi share vectors and hosts over a wide geographical area in Latin America. In this study, we propose a single molecular approach for simultaneous detection and typing of T. rangeli and T. cruzi.MethodsA restriction fragment length polymorphism analysis of the mitochondrial cytochrome oxidase II gene (COII-RFLP) using enzyme AluI and different amounts of DNA from the major genetic groups of T. rangeli and T. cruzi (KP1+/KP1- and DTU-I/DTU-II) was carried out. The same marker was tested on the other T. cruzi DTUs (DTU-III to DTU-VI) and on DNA extracted from gut contents of experimentally infected triatomines.ResultsThe COII PCR generates a ~400 bp fragment, which after digestion with AluI (COII-RFLP) can be used to distinguish T. rangeli from T. cruzi and simultaneously differentiate the major genetic groups of T. rangeli (KP1+ and KP1-) and T. cruzi (DTU-I and DTU-II). The COII-RFLP generated bands of ~120 bp and ~280 bp for KP1+, whereas for KP1- no amplicon cleavage was observed. For T. cruzi, digestion of COII revealed a ~300 bp band for DTU-I and a ~250 bp band for DTU-II. For DTU-III to DTU-VI, COII-RFLP generated bands ranging from ~310 to ~330 bp, but the differentiation of these DTUs was not as clear as the separation between DTU-I and DTU-II. After AluI digestion, a species-specific fragment of ~80 bp was observed for all DTUs of T. cruzi. No cross-amplification was observed for Leishmania spp., T. vivax or T. evansi.ConclusionsThe COII-RFLP allowed simultaneous detection and typing of T. rangeli and T. cruzi strains according to their major genetic groups (KP1+/KP1- and DTU-I/DTU-II) in vitro and in vivo, providing a reliable and sensitive tool for epidemiological studies in areas where T. rangeli and T. cruzi coexist.

Mixed infections with Trypanosoma cruzi and Trypanosoma rangeli and their different genetic groups occur frequently in vertebrate hosts and are difficult to detect by serology. In the present study, we evaluated the limit of detection of polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) analysis of cytochrome oxidase II (COII) for the identification of genetic groups of these two parasites in blood and tissue from vertebrate hosts. Reconstitution experiments were performed using human blood (TcI/TcII and KP1+/KP1-) and mouse tissue (TcI/TcII). We tested blood from patients who were in the chronic phase of Chagas disease and tissue from animals that were experimentally infected with all possible combinations of six discrete typing units. In blood samples, T. cruzi and T. rangeli were detected when 5 parasites (pa) were present in the sample, and genetic groups were identified when at least 50 pa were present in the sample. T. cruzi alone could be detected with 1 pa and genotyped (TcI/TcII) with 2 pa. T. rangeli was detected with 2 pa and genotyped (KP+/KP1-) with 25 pa. The present method more readily detected TcII and KP1- in both admixtures and alone. In mouse tissue, TcI and TcII were detected with at least 25 pa. The analysis of blood samples from patients and tissue from animals that were experimentally infected revealed low parasite loads in these hosts, which were below the limit of detection of the present method and could not be genotyped. Our findings indicate that the performance of PCR/RFLP analysis of COII is directly related to the amount and proportion of parasites that are present in the sample and the genetic groups to which the parasites belong.

Trypanosoma cruzi, the etiologic agent of Chagas disease (CD), is transmitted by hematophagous insects belonging to the subfamily Triatominae. After elimination of Triatoma infestans, the infestation of human dwellings by secondary species of vectors continues to pose a risk of transmission of the parasite. Our aim was to investigate the T. cruzi presence in triatomines and humans in rural households in the State of Paraná, southern Brazil. The capture of the insects was carried out by technicians of the municipalities after residents reported the outbreak. Five residents and 27 triatomines captured in four municipalities in the North and Midwest of the state were evaluated. The research of T. cruzi was carried out using parasitological, serological, and molecular techniques, in human blood, excreta, intestinal contents and insect macerate. Panstrongylus megistus, P. geniculatus and Triatoma sp.  were identified. Ten specimens of P. megistus were captured in a house in Mandaguari with five residents and presented an infection rate of 70% for T. cruzi like. All residents tested negative for T. cruzi infection. Another 15 P. megistus were captured in the peridomicile in Janiópolis and had 100.0% positivity. The only adult specimen of P. geniculatus captured in the intradomicile in Amaporã, as well as the nymph of Triatoma in the peridomicile in Paiçandu, were negative. The finding of P. megistus naturally infected by T. cruzi in households in rural area of Paraná demonstrates a potential risk of vector transmission of CD in these regions.