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Background: Trypanosoma cruzi, the etiological agent of Chagas Disease, exhibits remarkable genetic diversity and is classified into different Discrete Typing Units (DTUs). Strain typing techniques are crucial for studying T. cruzi, because their DTUs have significant biological differences from one another. However, there is currently no methodological strategy for the direct typing of biological materials that has sufficient sensitivity, specificity, and reproducibility. The high diversity and copy number of the minicircle hypervariable regions (mHVRs) makes it a viable target for typing., Methodology/principal Findings: Approximately 24 million reads obtained by amplicon sequencing of the mHVR were analyzed for 62 strains belonging to the six main T. cruzi DTUs. To build reference databases of mHVR diversity for each DTU and to evaluate this target as a typing tool. Strains of the same DTU shared more mHVR clusters than strains of different DTUs, and clustered together. Different identity thresholds were used to build the reference sets of the mHVR sequences (85% and 95%, respectively). The 95% set had a higher specificity and was more suited for detecting co-infections, whereas the 85% set was excellent for identifying the primary DTU of a sample. The workflow's capacity for typing samples obtained from cultures, a set of whole-genome data, under various simulated PCR settings, in the presence of co-infecting lineages and for blood samples was also assessed., Conclusions/significance: We present reference databases of mHVR sequences and an optimized typing workflow for T. cruzi including a simple online tool for deep amplicon sequencing analysis (https://ntomasini.github.io/cruzityping/). The results show that the workflow displays an equivalent resolution to that of the other typing methods. Owing to its specificity, sensitivity, relatively low cost, and simplicity, the proposed workflow could be an alternative for screening different types of samples., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Rusman et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Trypanosoma cruzi , as other kinetoplastids, has a complex mechanism of editing of mitochondrial mRNAs that requires guide RNAs (gRNAs) coded in DNA minicircles in the kinetoplast. There are many variations on this mechanism among species. mRNA editing and gRNA repertoires are almost unknown in T. cruzi . Here, gRNAs were inferred based on deep-sequenced minicircle hypervariable regions (mHVRs) and editing cascades were rebuilt in strains belonging to the six main T. cruzi lineages. Inferred gRNAs were clustered according to their sequence similarity to constitute gRNA classes. Extreme diversity of gRNA classes was observed, which implied highly divergent gRNA repertoires among different lineages, even within some lineages. In addition, a variable gRNA class redundancy (i.e., different gRNA classes editing the same mRNA region) was detected among strains. Some strains had upon four times more gRNA classes than others. Such variations in redundancy affected gRNA classes of all mRNAs in a concerted way, i.e., there are correlated variations in the number of gRNAs classes editing each mRNA. Interestingly, cascades were incomplete for components of the respiratory complex I in several strains. Finally, gRNA classes of different strains may potentially edit mitochondrial mRNAs from other lineages in the same way as they edit their own mitochondrial mRNAs, which is a prerequisite for biparental inheritance of minicircle in hybrids. We propose that genetic exchange and biparental inheritance of minicircles combined with minicircle drift due to (partial) random segregation of minicircles during kDNA replication is a suitable hypothesis to explain the divergences among strains and the high levels of gRNA redundancy in some strains. In addition, our results support that the complex I may not be required in some stages in the life cycle as previously shown and that linkage (in the same minicircle) of gRNAs that edit different mRNAs may prevent gRNA class lost in such stage., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Rusman, Floridia-Yapur, Tomasini and Diosque.)

Background: Genetic exchange in Trypanosoma cruzi is controversial not only in relation to its frequency, but also to its mechanism. Parasexual genetic exchange has been proposed based on laboratory hybrids, but population genomics strongly suggests meiosis in T. cruzi. In addition, mitochondrial introgression has been reported several times in natural isolates although its mechanism is not fully understood yet. Moreover, hybrid T. cruzi DTUs (TcV and TcVI) have inherited at least part of the kinetoplastic DNA (kDNA = mitochondrial DNA) from both parents., Methodology/principal Findings: In order to address such topics, we sequenced and analyzed fourteen nuclear DNA fragments and three kDNA maxicircle genes in three TcI stocks which are natural clones potentially involved in events of genetic exchange. We also deep-sequenced (a total of 6,146,686 paired-end reads) the minicircle hypervariable region (mHVR) of the kDNA in such three strains. In addition, we analyzed the DNA content by flow cytometry to address cell ploidy. We observed that most polymorphic sites in nuclear loci showed a hybrid pattern in one cloned strain and the other two cloned strains were compatible as parental strains (or nearly related to the true parents). The three clones had almost the same ploidy and the DNA content was similar to the reference strain Sylvio (a nearly diploid strain). Despite maxicircle genes evolve faster than nuclear housekeeping ones, we detected no polymorphisms in the sequence of three maxicircle genes showing mito-nuclear discordance. Lastly, the hybrid stock shared 66% of its mHVR clusters with one putative parent and 47% with the other one; in contrast, the putative parental stocks shared less than 30% of the mHVR clusters between them., Conclusions/significance: The results suggest a reductive division, a natural hybridization, biparental inheritance of the minicircles in the hybrid and maxicircle introgression. The models including such phenomena and explaining the relationships between these three clones are discussed., Competing Interests: The authors have declared that no competing interests exist.

The discovery and characterization of novel parasite antigens to improve the diagnosis of Trypanosoma cruzi by serological methods and for accurate and rapid follow-up of treatment efficiency are still needed. TcTASV is a T. cruzi -specific multigene family, whose products are expressed on the parasite stages present in the vertebrate host. In a previous work, a mix of antigens from subfamilies TcTASV-A and TcTASV-C (Mix A + C) was sensitive and specific to identify dogs with active infection of high epidemiological relevance. Here, TcTASV-A and TcTASV-C were assayed separately as well as together (Mix A + C) in an ELISA format on human samples. The Mix A + C presented moderate sensitivity (78%) but high diagnostic accuracy with a 100% of specificity, evaluated on healthy, leishmaniasic, and Strongyloides stercoralis infected patients. Moreover, antibody levels of pediatric patients showed-2 years posttreatment-diminished reactivity against the Mix A + C ( P < 0.0001), pointing TcTASV antigens as promising tools for treatment follow-up.

Background: Trypanosoma cruzi, the protozoan causative of Chagas disease, is classified into six main Discrete Typing Units (DTUs): TcI-TcVI. This parasite has around 105 copies of the minicircle hypervariable region (mHVR) in their kinetoplastic DNA (kDNA). The genetic diversity of the mHVR is virtually unknown. However, cross-hybridization assays using mHVRs showed hybridization only between isolates belonging to the same genetic group. Nowadays there is no methodologic approach with a good sensibility, specificity and reproducibility for direct typing on biological samples. Due to its high copy number and apparently high diversity, mHVR becomes a good target for typing., Methodology/principal Findings: Around 22 million reads, obtained by amplicon sequencing of the mHVR, were analyzed for nine strains belonging to six T. cruzi DTUs. The number and diversity of mHVR clusters was variable among DTUs and even within a DTU. However, strains of the same DTU shared more mHVR clusters than strains of different DTUs and clustered together. In addition, hybrid DTUs (TcV and TcVI) shared similar percentages (1.9-3.4%) of mHVR clusters with their parentals (TcII and TcIII). Conversely, just 0.2% of clusters were shared between TcII and TcIII suggesting biparental inheritance of the kDNA in hybrids. Sequencing at low depth (20,000-40,000 reads) also revealed 95% of the mHVR clusters for each of the analyzed strains. Finally, the method revealed good correlation in cluster identity and abundance between different replications of the experiment (r = 0.999)., Conclusions/significance: Our work sheds light on the sequence diversity of mHVRs at intra and inter-DTU level. The mHVR amplicon sequencing workflow described here is a reproducible technique, that allows multiplexed analysis of hundreds of strains and results promissory for direct typing on biological samples in a future. In addition, such approach may help to gain knowledge on the mechanisms of the minicircle evolution and phylogenetic relationships among strains., Competing Interests: The authors have declared that no competing interests exist.

Phylogenetic relationships among major lineages of Trypanosoma cruzi are still debatable. Particularly, it is controversial the origin of two main lineages: TcIII and TcIV. Some authors proposed that these lineages have been the result of an ancient hybridization between TcI and TcII, and this was one of the most accepted evolutionary models in the scientific community for several years. In the present paper we analyse several genomes of T. cruzi in order to examine if there is evidence supporting that TcIII is an ancient TcI/TcII hybrid. Our results show that TcIII is mainly related to TcI and not to TcII and there is few evidence of mosaicism for TcIII. Our results challenge the hypothesis of the ancient TcI/TcII hybridization., (Copyright © 2017 Elsevier B.V. All rights reserved.)

People living in areas with active vector-borne transmission of Chagas disease have multiple contacts with its causative agent, Trypanosoma cruzi. Reinfections by T. cruzi are possible at least in animal models leading to lower or even hardly detectable parasitaemia. In humans, although reinfections are thought to have major public health implications by increasing the risk of chronic manifestations of the disease, there is little quantitative knowledge about their frequency and the timing of parasite re-inoculation in the course of the disease. Here, we implemented stochastic agent-based models i) to estimate the rate of re-inoculation in humans and ii) to assess how frequent are reinfections during the acute and chronic stages of the disease according to alternative hypotheses on the adaptive immune response following a primary infection. By using a hybrid genetic algorithm, the models were fitted to epidemiological data of Argentinean rural villages where mixed infections by different genotypes of T. cruzi reach 56% in humans. To explain this percentage, the best model predicted 0.032 (0.008-0.042) annual reinfections per individual with 98.4% of them occurring in the chronic phase. In addition, the parasite escapes to the adaptive immune response mounted after the primary infection in at least 20% of the events of re-inoculation. With these low annual rates, the risks of reinfection during the typically long chronic stage of the disease stand around 14% (4%-18%) and 60% (21%-70%) after 5 and 30 years, with most individuals being re-infected 1-3 times overall. These low rates are better explained by the weak efficiency of the stercorarian mode of transmission than a highly efficient adaptive immune response. Those estimates are of particular interest for vaccine development and for our understanding of the higher risk of chronic disease manifestations suffered by infected people living in endemic areas.

Several different models of Trypanosoma cruzi evolution have been proposed. These models suggest that scarce events of genetic exchange occurred during the evolutionary history of this parasite. In addition, the debate has focused on the existence of one or two hybridisation events during the evolution of T. cruzi lineages. Here, we reviewed the literature and analysed available sequence data to clarify the phylogenetic relationships among these different lineages. We observed that TcI, TcIII and TcIV form a monophyletic group and that TcIII and TcIV are not, as previously suggested, TcI-TcII hybrids. Particularly, TcI and TcIII are sister groups that diverged around the same time that a widely distributed TcIV split into two clades (TcIVS and TcIVN). In addition, we collected evidence that TcIII received TcIVS kDNA by introgression on several occasions. Different demographic hypotheses (surfing and asymmetrical introgression) may explain the origin and expansion of the TcIII group. Considering these hypotheses, genetic exchange should have been relatively frequent between TcIII and TcIVS in the geographic area in which their distributions overlapped. In addition, our results support the hypothesis that two independent hybridisation events gave rise to TcV and TcVI. Consequently, TcIVS kDNA was first transferred to TcIII and later to TcV and TcVI in TcII/TcIII hybridisation events.

Many infectious diseases arise from co-infections or re-infections with more than one genotype of the same pathogen. These mixed infections could alter host fitness, the severity of symptoms, success in pathogen transmission and the epidemiology of the disease. Trypanosoma cruzi, the etiological agent of Chagas disease, exhibits a high biological variability often correlated with its genetic diversity. Here, we developed an experimental approach in order to evaluate biological interaction between three T. cruzi isolates belonging to different Discrete Typing Units (DTUs TcIII, TcV and TcVI). These isolates were obtained from a restricted geographical area in the Chaco Region. Different mixed infections involving combinations of two isolates (TcIII + TcV, TcIII + TcVI and TcV + TcVI) were studied in a mouse model. The parameters evaluated were number of parasites circulating in peripheral blood, histopathology and genetic characterization of each DTU in different tissues by DNA hybridization probes. We found a predominance of TcVI isolate in blood and tissues respect to TcIII and TcV; and a decrease of the inflammatory response in heart when the damage of mice infected with TcVI and TcIII + TcVI mixture were compared. In addition, simultaneous presence of two isolates in the same tissue was not detected. Our results show that biological interactions between isolates with different biological behaviors lead to changes in their biological properties. The occurrence of interactions among different genotypes of T. cruzi observed in our mouse model suggests that these phenomena could also occur in natural cycles in the Chaco Region.

The transmission cycles of Trypanosoma cruzi in the Gran Chaco are complex networks involving domestic and wild components, whose interrelationships are not well understood. Knowing the circuit of transmission of the different Discrete Typing Units (DTUs) of T. cruzi in the complex environment of the Chaco region is relevant to understanding how the different components (reservoirs, vectors, ecotopes) interact. In the present study we identified the DTUs infecting humans and dogs in two rural areas of the Gran Chaco in Argentina, using molecular methods which avoid parasite culture. Blood samples of humans and dogs were typified by PCR-DNA blotting and hybridization assays with five specific DNA probes (TcI, TcII, TcIII, TcV and TcVI). PCR analyses were performed on seropositive human and dog samples and showed the presence of T. cruzi DNA in 41.7% (98/235) and 53% (35/66) samples, respectively. The identification of infective DTUs was determined in 83.6% (82/98) and 91.4% (32/35) in human and dog samples, respectively. Single infections (36.7% - 36/98) and a previously not detected high proportion of mixed infections (47.9% - 47/98) were found. In a 15.3% (15/98) of samples the infecting DTU was not identified. Among the single infections TcV was the most prevalent DTU (30.6% - 30/98) in human samples; while TcVI (42.8% - 15/35) showed the highest prevalence in dog samples. TcV/TcVI was the most prevalent mixed infection in humans (32.6% - 32/98); and TcI/TcVI (14.3% - 5/35) in dogs. Significant associations between TcV with humans and TcVI with dogs were detected. For the first time, the presence of TcIII was detected in humans from this region. The occurrence of one human infected whit TcIII (a principally wild DTU) could be suggested the emergence of this, in domestic cycles in the Gran Chaco., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Trypanosoma cruzi has been historically classified as a species with preponderant clonal evolution (PCE). However, with the advent of highly polymorphic markers and studies at geographically reduced scales, the PCE in T. cruzi was challenged. In fact, some studies have suggested that recombination in T. cruzi lineage I (TcI) is much more frequent than previously believed. Further analyses of TcI populations from different geographical regions of Latin America are needed to examine this hypothesis. In the present study, we contribute to this topic by analyzing the population structure of TcI from a restricted geographical area in the Chaco region, Argentina. We analyzed TcI isolates from different hosts and vectors using a Multilocus Sequence Typing (MLST) approach. These isolates were previously characterized by sequencing the spliced leader intergenic region (SL-IR). Low levels of incongruence and well-supported clusters for MLST dataset were obtained from the analyses. Moreover, high linkage disequilibrium was found and five repeated and overrepresented genotypes were detected. In addition, a good correspondence between SL-IR and MLST was observed which is expected under PCE. However, recombination is not ruled out because five out of 28 pairs of loci were incompatible with strict clonality and one possible genetic exchange event was detected. Overall, our results represent evidence of PCE in TcI from the study area. Finally, considering our findings we discuss the scenario for the genetic structure of TcI., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Trypanosoma cruzi, the aetiological agent of Chagas disease possess extensive genetic diversity. This has led to the development of a plethora of molecular typing methods for the identification of both the known major genetic lineages and for more fine scale characterization of different multilocus genotypes within these major lineages. Whole genome sequencing applied to large sample sizes is not currently viable and multilocus enzyme electrophoresis, the previous gold standard for T. cruzi typing, is laborious and time consuming. In the present work, we present an optimized Multilocus Sequence Typing (MLST) scheme, based on the combined analysis of two recently proposed MLST approaches. Here, thirteen concatenated gene fragments were applied to a panel of T. cruzi reference strains encompassing all known genetic lineages. Concatenation of 13 fragments allowed assignment of all strains to the predicted Discrete Typing Units (DTUs), or near-clades, with the exception of one strain that was an outlier for TcV, due to apparent loss of heterozygosity in one fragment. Monophyly for all DTUs, along with robust bootstrap support, was restored when this fragment was subsequently excluded from the analysis. All possible combinations of loci were assessed against predefined criteria with the objective of selecting the most appropriate combination of between two and twelve fragments, for an optimized MLST scheme. The optimum combination consisted of 7 loci and discriminated between all reference strains in the panel, with the majority supported by robust bootstrap values. Additionally, a reduced panel of just 4 gene fragments displayed high bootstrap values for DTU assignment and discriminated 21 out of 25 genotypes. We propose that the seven-fragment MLST scheme could be used as a gold standard for T. cruzi typing, against which other typing approaches, particularly single locus approaches or systematic PCR assays based on amplicon size, could be compared.

The biological behavior of the different Trypanosoma cruzi strains is still unclear and the importance of exploring the relevance of these differences in natural isolates is of great significance. Herein we describe the biological behavior of four T. cruzi isolates circulating sympatrically in a restricted geographic area in Argentina endemic for Chagas Disease. These isolates were characterized as belonging to the Discrete Typing Units (DTUs) TcI, TcIII, TcV and TcVI as shown by Multilocus Enzyme Electrophoresis and Multilocus Sequence Typing. In order to study the natural behavior of the different isolates and to preserve their natural properties, we developed a vector transmission model that allows their maintenance in the laboratory. The model consisted of serial passages of these parasites between insect vectors and mice. Vector-derived parasite forms were then inoculated in C57BL/6J mice and number of parasite in peripheral blood, serological response and histological damage in acute and chronic phases of the infection were measured. Parasites from DTUs TcI, TcIII and TcVI were detected by direct fresh blood examination, while TcV parasites could only be detected by Polimerase Chain Reaction. No significant difference in the anti-T. cruzi antibody response was found during the chronic phase of infection, except for mice infected with TcV parasites where no antibodies could be detected. Histological sections showed that TcI isolate produced more damage in skeletal muscle while TcVI induced more inflammation in the heart. This work shows differential biological behavior among different parasite isolates obtained from the same cycle of transmission, permitting the opportunity to formulate future hypotheses of clinical and epidemiological importance., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A Multilocus Sequence Typing (MLST) scheme was designed and applied to a set of 20 Trypanosoma cruzi stocks belonging to three main discrete typing units (T. cruzi I, V and VI) from a geographically restricted Chagas disease endemic area in Argentina, 12 reference strains comprising two from each of the six main discrete typing units of the parasite (T. cruzi I-VI), and one T. cruzi marinkellei strain. DNA fragments (≅400-bp) from 10 housekeeping genes were sequenced. A total of 4178 bp were analyzed for each stock. In all, 154 polymorphic sites were identified. Ninety-five sites were heterozygous in at least one analyzed stock. Seventeen diploid sequence types were identified from 32 studied T. cruzi stocks (including the reference strains). All stocks were correctly assigned to their corresponding discrete typing units. We propose this MLST scheme as provisional, with scope for improvement by studying new gene targets on a more diverse sample of stocks, in order to define an optimized MLST scheme for T. cruzi. This approach is an excellent candidate to become the gold standard for T. cruzi genetic typing. We suggest that MLST will have a strong impact on molecular epidemiological studies of Chagas disease and the phylogenetics of its causative agent., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Internal and geographical clustering within Trypanosoma cruzi I (TcI) has been recently revealed by using Multilocus Microsatellite Typing and sequencing of the Spliced-Leader Intergenic Region (SL-IR). In the present work, 14 isolates and 11 laboratory-cloned stocks obtained from a geographically restricted area in Chaco Province, Argentina, were analyzed by PCR and sequencing of SL-IR. We were able to differentiate 8 different genotypes that clustered into 4 groups. One of these groups was classified within the formerly described haplotype A and another one within the recently described SL-IR group E. Both were phylogenetically well-supported. In contrast, none of the stocks from the Chaco province were grouped within the cluster previously named haplotype D despite the fact that they shared a similar microsatellite motif in the SL-IR. No evidence of recombination or gene conversion within these stocks was found. On the other hand, multiple ambiguous alignments in the microsatellite region of SL-IR, affecting the tree topology and relationships among groups were detected. Finally, since there are multiple copies of the SL-IR, and they are arranged in tandem, we discuss how molecular processes affecting this kind of sequences could mislead phylogenetic inference., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The intergenic region of spliced-leader (SL-IR) genes from 105 Trypanosoma cruzi I (Tc I) infected biological samples, culture isolates and stocks from 11 endemic countries, from Argentina to the USA were characterised, allowing identification of 76 genotypes with 54 polymorphic sites from 123 aligned sequences. On the basis of the microsatellite motif proposed by Herrera et al. (2007) to define four haplotypes in Colombia, we could classify these genotypes into four distinct Tc I SL-IR groups, three corresponding to the former haplotypes Ia (11 genotypes), Ib (11 genotypes) and Id (35 genotypes); and one novel group, Ie (19 genotypes). Genotypes harbouring the Tc Ic motif were not detected in our study. Tc Ia was associated with domestic cycles in southern and northern South America and sylvatic cycles in Central and North America. Tc Ib was found in all transmission cycles from Colombia. Tc Id was identified in all transmission cycles from Argentina and Colombia, including Chagas cardiomyopathy patients, sylvatic Brazilian samples and human cases from French Guiana, Panama and Venezuela. Tc Ie gathered five samples from domestic Triatoma infestans from northern Argentina, nine samples from wild Mepraia spinolai and Mepraia gajardoi and two chagasic patients from Chile and one from a Bolivian patient with chagasic reactivation. Mixed infections by Tc Ia+Tc Id, Tc Ia+Tc Ie and Tc Id+Tc Ie were detected in vector faeces and isolates from human and vector samples. In addition, Tc Ia and Tc Id were identified in different tissues from a heart transplanted Chagas cardiomyopathy patient with reactivation, denoting histotropism. Trypanosoma cruzi I SL-IR genotypes from parasites infecting Triatoma gerstaeckeri and Didelphis virginiana from USA, T. infestans from Paraguay, Rhodnius nasutus and Rhodnius neglectus from Brazil and M. spinolai and M. gajardoi from Chile are to our knowledge described for the first time., (Copyright © 2010 Australian Society for Parasitology Inc. All rights reserved.)

The model of predominant clonal evolution (PCE) proposed for micropathogens does not state that genetic exchange is totally absent, but rather, that it is too rare to break the prevalent PCE pattern. However, the actual impact of this “residual” genetic exchange should be evaluated. Multilocus Sequence Typing (MLST) is an excellent tool to explore the problem. Here, we compared online available MLST datasets for seven eukaryotic microbial pathogens: Trypanosoma cruzi, the Fusarium solani complex, Aspergillus fumigatus, Blastocystis subtype 3, the Leishmania donovani complex, Candida albicans and Candida glabrata. We first analyzed phylogenetic relationships among genotypes within each dataset. Then, we examined different measures of branch support and incongruence among loci as signs of genetic structure and levels of past recombination. The analyses allow us to identify three types of genetic structure. The first was characterized by trees with well-supported branches and low levels of incongruence suggesting well-structured populations and PCE. This was the case for the T. cruzi and F. solani datasets. The second genetic structure, represented by Blastocystis spp., A. fumigatus and the L. donovani complex datasets, showed trees with weakly-supported branches but low levels of incongruence among loci, whereby genetic structuration was not clearly defined by MLST. Finally, trees showing weakly-supported branches and high levels of incongruence among loci were observed for Candida species, suggesting that genetic exchange has a higher evolutionary impact in these mainly clonal yeast species. Furthermore, simulations showed that MLST may fail to show right clustering in population datasets even in the absence of genetic exchange. In conclusion, these results make it possible to infer variable impacts of genetic exchange in populations of predominantly clonal micro-pathogens. Moreover, our results reveal different problems of MLST to determine the genetic structure in these organisms that should be considered. [ABSTRACT FROM AUTHOR]