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Trypanosoma cruzi, the causative agent of Chagas disease, presents wide genetic diversity. Currently, six discrete typing units (DTUs), named TcI to TcVI, and a seventh one called TcBat are used for strain typing. Beyond the debate concerning this classification, this systematic review has attempted to provide an inventory by compiling the results of 137 articles that have used it. A total of 6,343 DTU identifications were analyzed according to the geographical and host origins. Ninety-one percent of the data available is linked to South America. This sample, although not free of potential bias, nevertheless provides today's picture of T. cruzi genetic diversity that is closest to reality. DTUs were genotyped from 158 species, including 42 vector species. Remarkably, TcI predominated in the overall sample (around 60%), in both sylvatic and domestic cycles. This DTU known to present a high genetic diversity, is very widely distributed geographically, compatible with a long-term evolution. The marsupial is thought to be its most ancestral host and the Gran Chaco region the place of its putative origin. TcII was rarely sampled (9.6%), absent, or extremely rare in North and Central America, and more frequently identified in domestic cycles than in sylvatic cycles. It has a low genetic diversity and has probably found refuge in some mammal species. It is thought to originate in the south-Amazon area. TcIII and TcIV were also rarely sampled. They showed substantial genetic diversity and are thought to be composed of possible polyphyletic subgroups. Even if they are mostly associated with sylvatic transmission cycles, a total of 150 human infections with these DTUs have been reported. TcV and TcVI are clearly associated with domestic transmission cycles. Less than 10% of these DTUs were identified together in sylvatic hosts. They are thought to originate in the Gran Chaco region, where they are predominant and where putative parents exist (TcII and TcIII). Trends in host-DTU specificities exist, but generally it seems that the complexity of the cycles and the participation of numerous vectors and mammal hosts in a shared area, maintains DTU diversity.

Trypanosoma cruzi is a single-celled eukaryotic parasite responsible for Chagas disease, a major cause of morbidity and mortality in Central and South America. While the host-pathogen interactions of T. cruzi have been extensively studied in vertebrate models, investigations into its interactions within its insect host remain limited. To address this gap and establish a genetically tractable system for studying parasite-vector dynamics, we conducted quantitative kinetic infection studies using the Y strain of T. cruzi and the model vector Rhodnius prolixus . We began by comparing parasite infection kinetics from two genetically diverse strains of T. cruzi, Brazil and Y, and demonstrated that ingested parasites from both strains transiently expand in the anterior regions of the insect digestive tract with stable colonization occurring in the hindgut over the long term. Notably, we demonstrated that the clonal Y strain, contrary to previous reports, can effectively infect and persist across multiple developmental stages of R. prolixus . Additionally, comparison of movement of parasites versus inert fluorescent microspheres introduced into artificial blood meals suggests that T. cruzi colonization of the R. prolixus gut occurs passively through peristaltic movement during digestion, rather than through active parasite-mediated chemotaxis. These findings highlight the T. cruzi Y strain - R. prolixus model system as a promising tool for the in-depth molecular characterization of parasite-vector interactions, potentially offering new insights into the biology of this neglected and deadly human pathogen.

Trypanosomatids of the Schizotrypanum clade display high ecological and evolutionary diversity, yet their sylvatic cycles remain understudied in many Neotropical biomes. We conducted the first integrative molecular survey of Trypanosoma diversity in a conserved seasonal tropical forest in the Yucatán Peninsula, Mexico. We screened Triatoma dimidiata vectors and small mammal hosts (rodents and bats) using satellite DNA (SatDNA) for Trypanosoma cruzi detection, Miniexon PCR for T. cruzi discrete typing unit (DTU) identification, and 18 S rRNA sequencing to identify other Trypanosoma species in bats. Trypanosoma cruzi was detected by SatDNA in 81.8% of collected vectors (95% CI: 75.07–87.38%; n  = 165) and in 46.6% of rodents (95% CI: 36.7–56.7%, n  = 103) and in 46.2% of bats (95% CI: 38.56–53.97%; n  = 171). Discrete typing unit (DTU) analysis revealed that T. cruzi DTU IV (TcIV) predominated in the conserved tropical forest, being mainly associated with vectors and rodents, whereas TcI was more frequent in bats. Phylogenetic reconstruction based on 18 S rRNA revealed multiple bat-associated lineages, including sequences related to T. wauwau , T. madeirae , and Neobat clades—reported here for the first time in Mexico. Our results underscore the role of protected forests as reservoirs of trypanosomatid diversity and the relevance of multi-host surveillance to better understand transmission dynamics. This study contributes novel insights into the ecology, biogeography, and evolutionary history of Trypanosoma spp. in Mesoamerica and supports efforts to strengthen early detection of potential zoonotic threats. Graphical Abstract Schematic representation of Trypanosoma cruzi discrete typing unit (DTU) associations in a sylvatic transmission cycle within a conserved seasonal tropical forest in Yucatán, Mexico. TcI (blue) was predominantly associated with bats (highly mobile hosts), while TcIV (purple) was primarily detected in rodents and triatomine vectors (low-dispersal hosts), illustrating distinct host-pathogen interaction patterns that reflect ecological filtering of T. cruzi lineages.

The transmission dynamics of Trypanosoma cruzi in natural environments exhibit considerable variation at the micro-locality scale. However, the specific biotic and abiotic factors driving this heterogeneity remain largely unidentified. The Atlantic Forest of the state of Espírito Santo (ES) presents a unique transmission network of T. cruzi , in which Triatoma vitticeps represents the absolute majority among existing triatomines, with high infection rates and diversity of genotypes, frequently invading homes. No infection was found in peridomestic mammals. This study aimed to elucidate the spatial and environmental distribution patterns of T. vitticeps and its infection by T. cruzi DTUs throughout Espírito Santo, quantifying the influence of abiotic variables on both vector occurrence and infection dynamics. Species Distribution Modeling (SDM) of T. cruzi genotypes in T. vitticeps collected in the Atlantic Forest of Espírito Santo was performed using the ModleR package, in the R programming language, with climate and landscape variables (~1km²) selected by Spearman’s correlation [-0.7 ≤ ρ ≤ 0.7]. True Skill Statistic (≥ 0.7) was used to evaluate model performance. Decision tree to classify T. vitticeps infection by T. cruzi was created using machine learning algorithms in WEKA 3.8.6 software. The SDMs of T. vitticeps and its infection demonstrated: i. Central and South mesoregions presented better environmental conditions for their occurrence; ii. association with mountainous regions with high altitudes, humid and superhumid, with vegetation density and vigor and high values of topographic diversity; iii. Schoener similarity suggests Z3 is mixed, dominated by TcIV and TcIII in Central–South, with TcIII influence Northwest and North Coast; iv. Infection was explained by wind speed, mammal richness, and temperature, with the decision tree identifying 84% of positives and 29% of negatives. T. vitticeps may originate in high-altitude regions and disperse via wind to lowlands, promoting domiciliary invasion and supporting previously hypothesized long-distance transmission of T. cruzi .

Background Trypanosoma conorhini and Trypanosoma rangeli , like Trypanosoma cruzi, are kinetoplastid protist parasites of mammals displaying divergent hosts, geographic ranges and lifestyles. Largely nonpathogenic T. rangeli and T. conorhini represent clades that are phylogenetically closely related to the T. cruzi and T. cruzi -like taxa and provide insights into the evolution of pathogenicity in those parasites. T. rangeli , like T. cruzi is endemic in many Latin American countries, whereas T. conorhini is tropicopolitan. T. rangeli and T. conorhini are exclusively extracellular, while T. cruzi has an intracellular stage in the mammalian host. Results Here we provide the first comprehensive sequence analysis of T. rangeli AM80 and T. conorhini 025E, and provide a comparison of their genomes to those of T. cruzi G and T. cruzi CL, respectively members of T. cruzi lineages TcI and TcVI. We report de novo assembled genome sequences of the low-virulent T. cruzi G, T. rangeli AM80, and T. conorhini 025E ranging from ~ 21–25 Mbp, with ~ 10,000 to 13,000 genes, and for the highly virulent and hybrid T. cruzi CL we present a ~ 65 Mbp in-house assembled haplotyped genome with ~ 12,500 genes per haplotype. Single copy orthologs of the two T. cruzi strains exhibited ~ 97% amino acid identity, and ~ 78% identity to proteins of T. rangeli or T. conorhini . Proteins of the latter two organisms exhibited ~ 84% identity. T. cruzi CL exhibited the highest heterozygosity. T. rangeli and T. conorhini displayed greater metabolic capabilities for utilization of complex carbohydrates, and contained fewer retrotransposons and multigene family copies, i.e. trans-sialidases, mucins, DGF-1, and MASP, compared to T. cruzi . Conclusions Our analyses of the T. rangeli and T. conorhini genomes closely reflected their phylogenetic proximity to the T. cruzi clade, and were largely consistent with their divergent life cycles. Our results provide a greater context for understanding the life cycles, host range expansion, immunity evasion, and pathogenesis of these trypanosomatids.

Chagas disease (CD), caused by infection with the protozoan parasite Trypanosoma cruzi, is a neglected tropical illness that affects 6-7 million individuals worldwide. CD may progress to chronic cardiac (CARD), digestive (DIG), or cardio-digestive (CARD/DIG) forms. Parasite genetic diversity, defined in discrete typing units (DTUs) TcI-TcVI and TcBat, may contribute to clinical forms. Here, we challenged this idea by studying a population born in the State of Pernambuco, Northeast of Brazil. Patients born in eco-geographically distinct mesoregions (Sertão, Agreste, Zona da Mata, and Metropolitan Region) attending the referral PROCAPE hospital were serologically diagnosed for CD and characterized as non-CARD (16.5%), CARD (67%), DIG (12.1%), and CARD/DIG (4.3%). Out of 346 CD patients not subjected to etiological treatment, 128 (37%) were positive for conventional PCR targeting T. cruzi kDNA. DTU genotyping using pre-established primers and algorithms revealed 85/128 (66%) samples amplified for at least one target, and of these, 49 (58%) were classified into DTUs, showing a higher distribution of DTUs TcV (16) and TcIII (13) but also TcIV (7), TcI (3), TcVI (3), and TcII (1). Mixed infections by TcI + TcV (2), TcIII + TcIV (3), and TcIII + TcV (1) were also found. Out of the 49 classified samples, 36 were CARD patients (73.5%), infected mainly by TcV (13) and TcIII (10). Considering patients' birthplace, in the Agreste, all DTUs were detected, with prevailing TcV and TcIII; in Zona da Mata, DTU characterization reflected the input of samples (21/49, 43%), again mainly TcV and TcIII. DTUs TcI-TcVI were only detected in the blood of patients born in the Agreste, area of ecological transition. Although not frequently found in human infections in Brazil, the prevailing TcV, TcIII, and TcIV were detected in patients born in Pernambuco. These data could not disclose an identifiable association of T. cruzi DTU circulating in the blood of patients with clinical forms of CD.

Background The DNA barcoding system using the cytochrome c oxidase subunit 1 mitochondrial gene ( cox 1 or COI ) is highly efficient for discriminating vertebrate and invertebrate species. In the present study, we examined the suitability of cox 1 as a marker for Trypanosoma cruzi identification from other closely related species . Additionally, we combined the sequences of cox 1 and the nuclear gene glucose-6-phosphate isomerase ( GPI ) to evaluate the occurrence of mitochondrial introgression and the presence of hybrid genotypes. Methods Sixty-two isolates of Trypanosoma spp. obtained from five of the six Brazilian biomes (Amazon Forest, Atlantic Forest, Caatinga, Cerrado and Pantanal) were sequenced for cox 1 and GPI gene fragments. Phylogenetic trees were reconstructed using neighbor-joining, maximum likelihood, parsimony and Bayesian inference methods. Molecular species delimitation was evaluated through pairwise intraspecific and interspecific distances, Automatic Barcode Gap Discovery, single-rate Poisson Tree Processes and multi-rate Poisson Tree Processes. Results Both cox 1 and GPI genes recognized and differentiated T. cruzi , Trypanosoma cruzi marinkellei , Trypanosoma dionisii and Trypanosoma rangeli . Cox 1 discriminated Tcbat, TcI, TcII, TcIII and TcIV. Additionally, TcV and TcVI were identified as a single group. Cox 1 also demonstrated diversity in the discrete typing units (DTUs) TcI, TcII and TcIII and in T. c. marinkellei and T. rangeli . Cox 1 and GPI demonstrated TcI and TcII as the most genetically distant branches, and the position of the other T. cruzi DTUs differed according to the molecular marker. The tree reconstructed with concatenated cox 1 and GPI sequences confirmed the separation of the subgenus Trypanosoma ( Schizotrypanum ) sp. and the T. cruzi DTUs TcI, TcII, TcIII and TcIV. The evaluation of single nucleotide polymorphisms (SNPs) was informative for DTU differentiation using both genes. In the cox 1 analysis, one SNP differentiated heterozygous hybrids from TcIV sequences. In the GPI analysis one SNP discriminated Tcbat from TcI, while another SNP distinguished TcI from TcIII. Conclusions DNA barcoding using the cox 1 gene is a reliable tool to distinguish T. cruzi from T. c. marinkellei , T. dionisii and T. rangeli and identify the main T. cruzi genotypes.

Chagas disease, caused by the hemoflagellate parasite Trypanosoma cruzi , is a major public health problem in Latin America due to its high prevalence and significant morbidity and mortality. The T. cruzi is classified into seven distinct genetic groups (TcI-TcVI) and TcBat, know as Discrete Typing Units (DTUs). Understanding DTU diversity is essential for improving diagnostic and therapeutic strategies and strengthening epidemiological surveillance. This study aimed to investigate the genetic diversity of T. cruzi in individuals with chronic Chagas disease from endemic municipalities in Northern Minas Gerais and the Jequitinhonha Valley, Brazil. It also evaluated associations between DTUs and age, parasitic load, antibody levels, and cardiac disease severity in participants from the SaMi-Trop cohort. Molecular typing was performed using conventional multilocus PCR directly from peripheral blood samples of individuals with the chronic cardiac form. Of the 80 patients included in the study, T. cruzi genotyping was performed in 37 samples (46.25%). Among the samples in which a single DTU was identified, TcI and TcVI presented equal frequencies (n = 5, 13.51% each). TcII was identified in four samples (n = 4, 10.81%), while TcV was identified in three samples (n = 3, 8.10%). Furthermore, three individuals presented mixed infections: TcV + TcVI, TcI + TcV + TcII/TcVI, and TcV + TcII. The highest frequency was observed for TcII/TcVI (n = 17, 45.94%), a classification that does not represent mixed infection. For statistical analysis, TcII/TcVI samples were classified as TcII. No significant differences were found between the DTUs and age or parasitic load. However, a wide variation in the average parasitic load was observed among individuals, ranging from 0.06 to 667 parasite equivalents/mL. An increase in anti- T. cruzi antibody titers was also observed. The findings of this study demonstrate the genetic diversity of the parasite in individuals with chronic cardiac Chagas disease and its distribution in a highly endemic region.

We sequenced maxicircles from T . cruzi strains representative of the species evolutionary diversity by using long-read sequencing, which allowed us to uncollapse their repetitive regions, finding that their real lengths range from 35 to 50 kb. T . cruzi maxicircles have a common architecture composed of four regions: coding region (CR), AT-rich region, short (SR) and long repeats (LR). Distribution of genes, both in order and in strand orientation are conserved, being the main differences the presence of deletions affecting genes coding for NADH dehydrogenase subunits, reinforcing biochemical findings that indicate that complex I is not functional in T . cruzi . Moreover, the presence of complete minicircles into maxicircles of some strains lead us to think about the origin of minicircles. Finally, a careful phylogenetic analysis was conducted using coding regions of maxicircles from up to 29 strains, and 1108 single copy nuclear genes from all of the DTUs, clearly establishing that taxonomically T . cruzi is a complex of species composed by group 1 that contains clades A (TcI), B (TcIII) and D (TcIV), and group 2 (1 and 2 do not coincide with groups I and II described decades ago) containing clade C (TcII), being all hybrid strains of the BC type. Three variants of maxicircles exist in T . cruzi : a, b and c, in correspondence with clades A, B, and C from mitochondrial phylogenies. While A and C carry maxicircles a and c respectively, both clades B and D carry b maxicircle variant; hybrid strains also carry the b- variant. We then propose a new nomenclature that is self-descriptive and makes use of both the phylogenetic relationships and the maxicircle variants present in T . cruzi .

Chagas disease, caused by the vector-borne protozoan Trypanosoma cruzi, is increasingly recognized in the southern U.S. Government-owned working dogs along the Texas-Mexico border could be at heightened risk due to prolonged exposure outdoors in habitats with high densities of vectors. We quantified working dog exposure to T. cruzi, characterized parasite strains, and analyzed associated triatomine vectors along the Texas-Mexico border. In 2015-2016, we sampled government working dogs in five management areas plus a training center in Texas and collected triatomine vectors from canine environments. Canine serum was tested for anti-T. cruzi antibodies with up to three serological tests including two immunochromatographic assays (Stat-Pak and Trypanosoma Detect) and indirect fluorescent antibody (IFA) test. The buffy coat fraction of blood and vector hindguts were tested for T. cruzi DNA and parasite discrete typing unit was determined. Overall seroprevalence was 7.4 and 18.9% (n = 528) in a conservative versus inclusive analysis, respectively, based on classifying weakly reactive samples as negative versus positive. Canines in two western management areas had 2.6-2.8 (95% CI: 1.0-6.8 p = 0.02-0.04) times greater odds of seropositivity compared to the training center. Parasite DNA was detected in three dogs (0.6%), including TcI and TcI/TcIV mix. Nine of 20 (45%) T. gerstaeckeri and T. rubida were infected with TcI and TcIV; insects analyzed for bloodmeals (n = 11) fed primarily on canine (54.5%). Government working dogs have widespread exposure to T. cruzi across the Texas-Mexico border. Interpretation of sample serostatus was challenged by discordant results across testing platforms and very faint serological bands. In the absence of gold standard methodologies, epidemiological studies will benefit from presenting a range of results based on different tests/interpretation criteria to encompass uncertainty. Working dogs are highly trained in security functions and potential loss of duty from the clinical outcomes of infection could affect the work force and have broad consequences.