Presence of Trypanosoma cruzi TcI and Trypanosoma dionisii in sylvatic bats from Yucatan, Mexico.

BACKGROUND: Chagas disease is caused by Trypanosoma cruzi, whose genetic structure is divided into six discrete typing units (DTUs) known as TcI-TcVI. In the Yucatan Peninsula, Mexico, information regarding the DTUs circulating in wild mammals is scarce, while this is important knowledge for our understanding of T. cruzi transmission dynamics. METHODS: In the current study, we sampled wild mammals in a sylvatic site of the Yucatan Peninsula and assessed their infection with T. cruzi by PCR. Then, for infected mammals, we amplified and sequenced nuclear and mitochondrial T. cruzi genetic markers for DTU identification. RESULTS: In total, we captured 99 mammals belonging to the orders Chiroptera, Rodentia and Didelphimorphia. The prevalence of infection with T. cruzi was 9% (9/99; 95% CI [5, 16]), and we identified TcI in a Jamaican fruit bat, Artibeus jamaicensis. Moreover, we fortuitously identified Trypanosoma dionisii in another Jamaican fruit bat and detected an unidentified Trypanosoma species in a third specimen. While the latter discoveries were not expected because we used primers designed for T. cruzi, this study is the first to report the identification of T. dionisii in a bat from Yucatan, Mexico, adding to a recent first report of T. dionisii in bats from Veracruz, and first report of this Trypanosoma species in Mexico. CONCLUSION: Further research is needed to enhance our knowledge of T. cruzi DTUs and Trypanosoma diversity circulating in wildlife in Southeastern Mexico.

Wide reference databases for typing Trypanosoma cruzi based on amplicon sequencing of the minicircle hypervariable region.

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.

Revisiting gene typing and phylogeny of Trypanosoma cruzi reference strains: Comparison of the relevance of mitochondrial DNA, single-copy nuclear DNA, and the intergenic region of mini-exon gene.

Chagas disease is a widespread neglected disease in Latin America. Trypanosoma cruzi, the causative agent of the disease, is currently subdivided into six DTUs (discrete typing units) named TcI-TcVI, and although no clear association has been found between parasite genetics and different clinical outcomes of the disease or different transmission cycles, genetic characterization of T. cruzi strains remains crucial for integrated epidemiological studies. Numerous markers have been used for this purpose, although without consensus. These include mitochondrial genes, single or multiple-copy nuclear genes, ribosomal RNA genes, and the intergenic region of the repeated mini-exon gene. To increase our knowledge of these gene sequences and their usefulness for strain typing, we sequenced fragments of three mitochondrial genes, nine single-copy nuclear genes, and the repeated intergenic part of the mini-exon gene by Next Generation Sequencing (NGS) on a sample constituted of 16 strains representative of T. cruzi genetic diversity, to which we added the corresponding genetic data of the 38 T. cruzi genomes fully sequenced until 2022. Our results show that single-copy nuclear genes remain the gold standard for characterizing T. cruzi strains; the phylogenetic tree from concatenated genes (3959 bp) confirms the six DTUs previously recognized and provides additional information about the alleles present in the hybrid strains. In the tree built from the three mitochondrial concatenated genes (1274 bp), three main clusters are identified, including one with TcIII, TcIV, TcV, and TcVI DTUs which are not separated. Nevertheless, mitochondrial markers remain necessary for detecting introgression and heteroplasmy. The phylogenetic tree built from the sequence alignment of the repeated mini-exon gene fragment (327 bp) displayed six clusters, but only TcI was associated with a single cluster. The sequences obtained from strains belonging to the other DTUs were scattered into different clusters. Therefore, while the mini-exon marker may bring, for some biological samples, some advantages in terms of sensibility due to its repeated nature, mini-exon sequences must be used with caution and, when possible, avoided for T. cruzi typing and phylogenetic studies.

Deep learning and wing interferential patterns identify Anopheles species and discriminate amongst Gambiae complex species.

We present a new and innovative identification method based on deep learning of the wing interferential patterns carried by mosquitoes of the Anopheles genus to classify and assign 20 Anopheles species, including 13 malaria vectors. We provide additional evidence that this approach can identify Anopheles spp. with an accuracy of up to 100% for ten out of 20 species. Although, this accuracy was moderate (> 65%) or weak (50%) for three and seven species. The accuracy of the process to discriminate cryptic or sibling species is also assessed on three species belonging to the Gambiae complex. Strikingly, An. gambiae, An. arabiensis and An. coluzzii, morphologically indistinguishable species belonging to the Gambiae complex, were distinguished with 100%, 100%, and 88% accuracy respectively. Therefore, this tool would help entomological surveys of malaria vectors and vector control implementation. In the future, we anticipate our method can be applied to other arthropod vector-borne diseases.

Molecular Characterization of Four Mexican Isolates of Trypanosoma cruzi and Their Profile Susceptibility to Nifurtimox.

PURPOSE: The objective of this study was to molecularly characterize Mexican isolates of T. cruzi obtained from infected triatomine bugs (the vectors of T. cruzi) and to evaluate their susceptibility to Nifurtimox (NFX). METHODS: Three isolates obtained from Triatoma dimidiata (collected in the State of Veracruz) and one isolate obtained from Triatoma bassolsae (collected in the State of Puebla) were molecularly characterized and the expression of genes associated with natural resistance to NFX was analyzed by qPCR. RESULTS: Molecular characterization by PCR showed that isolates Zn3, Zn5, and SRB1 belong to the DTU TcI, while isolate Sum3 belongs to TcIV. The latter was also confirmed by sequencing of mitochondrial genes. Isolate Zn5 was the most sensitive to treatment with NFX (IC50, 6.8 µM), isolates SRB1 and Zn3 were partially resistant (IC50, 12.8 µM and 12.7 µM) and isolate Sum3 showed a high degree of resistance to NFX (IC50, 21.4 µM). We also found an association between decreased NTR1 or OYE gene expression with NFX resistance. CONCLUSION: Our results also evidenced a high variability in the susceptibility to NFX of these T. cruzi isolates Central and Southeastern Mexico, suggesting the presence of naturally resistant isolates circulating in the country. These results have important implications for defining treatment policies for patients with Chagas disease.

In vitro susceptibility of Trypanosoma cruzi discrete typing units (DTUs) to benznidazole: A systematic review and meta-analysis.

BACKGROUND: Chagas disease, a neglected tropical disease endemic to Latin America caused by the parasite Trypanosoma cruzi, currently affects 6-7 million people and is responsible for 12,500 deaths each year. No vaccine exists at present and the only two drugs currently approved for the treatment (benznidazole and nifurtimox), possess serious limitations, including long treatment regimes, undesirable side effects, and frequent clinical failures. A link between parasite genetic variability and drug sensibility/efficacy has been suggested, but remains unclear. Therefore, we investigated associations between T. cruzi genetic variability and in vitro benznidazole susceptibility via a systematic article review and meta-analysis. METHODOLOGY/PRINCIPAL FINDINGS: In vitro normalized benznidazole susceptibility indices (LC50 and IC50) for epimastigote, trypomastigote and amastigote stages of different T. cruzi strains were recorded from articles in the scientific literature. A total of 60 articles, which include 189 assays, met the selection criteria for the meta-analysis. Mean values for each discrete typing unit (DTU) were estimated using the meta and metaphor packages through R software, and presented in a rainforest plot. Subsequently, a meta-regression analysis was performed to determine differences between estimated mean values by DTU/parasite stage/drug incubation times. For each parasite stage, some DTU mean values were significantly different, e.g. at 24h of drug incubation, a lower sensitivity to benznidazole of TcI vs. TcII trypomastigotes was noteworthy. Nevertheless, funnel plots detected high heterogeneity of the data within each DTU and even for a single strain. CONCLUSIONS/SIGNIFICANCE: Several limitations of the study prevent assigning DTUs to different in vitro benznidazole sensitivity groups; however, ignoring the parasite's genetic variability during drug development and evaluation would not be advisable. Our findings highlight the need for establishment of uniform experimental conditions as well as a screening of different DTUs during the optimization of new drug candidates for Chagas disease treatment.

Molecular data supports monophyly of Triatoma dispar complex within genus Triatoma.

The genus Triatoma contains numerous species, principal or secondary vectors of Chagas disease, which have been included in the three main lineages of Triatomini tribe based on morphological and biogeographical characteristics: North American, South American, and T. dispar complex. The three members of the T. dispar complex are distributed in Ecuador. This complex has been scarcely studied through molecular approaches, and the taxonomic position of this complex is not confirmed. In this study, we explored the phylogenetic relationships within the genus Triatoma, including five species from North and Central America, six from South America, and the three species belonging to the T. dispar complex. Partial sequences of four mitochondrial genes (Cyt b, COII, 16S-rRNA, 12S-rRNA) and two nuclear genes (18S-rRNA, ITS2) were obtained from 74 specimens. Phylogenetic trees were built with concatenated and single sequences through maximum likelihood (ML), maximum parsimony (MP), and Bayesian methods. The trees built using concatenated sequences showed three main branches (clusters) highly supported by significant bootstrap values; the T. dispar complex appeared as a monophyletic group separate from species of North and Central American origin and South American origin. On the contrary, for each gene tree, the three main clusters were not always significantly supported, mostly because genetic information is dramatically reduced when a single gene is considered. Consequently, concatenation of genes gives relevant results and is highly recommended for further in-depth examination of the relationships of several species and complexes of triatomines that remain unresolved. Moreover, our current molecular data fully revealed the division of genus Triatoma into at least three main genetic groups.

Would tropical climatic variations impact the genetic variability of triatomines: Rhodnius ecuadoriensis, principal vector of Chagas disease in Ecuador?

Rhodnius ecuadoriensis is one of the most important vector species of Chagas disease in Ecuador. This species is distributed in the Central coast region and in the south Andean region, and an incipient speciation process between these geographical populations was previously proposed. The current population genetics study only focused on the Central coast region and analyzed 96 sylvatic specimens of R. ecuadoriensis associated with Phytelephas aequatorialis palm trees. We used Cytb and 16S-rRNA sequences and a Cytb-16S-rRNA concatenated set to explore (i) the genetic variability, spatial structuring, and demographic history of R. ecuadoriensis, and to determine (ii) the relationship between the genetic and climatic variabilities. A particularly high genetic variability was observed without detectable general genetic structure; only some terminal genetic clusters were observed. We did not observe isolation by geographical distance (IBD), and it is likely that ancient expansion occurred, according to Fs index and mismatch distribution for Cytb-16S-rRNA concatenated sequences. Hierarchical clustering showed that the current locality origins of the bugs were grouped into four bioclimatic clusters. Genetic and bioclimatic distances were not correlated, but some genetic clusters were associated with bioclimatic ones. The results showed an ancient evolution of the species in the region with a possible old expansion. The absence of spatial genetic structure could be due to climatic conditions (possible selection of singular genotypes) and to passive transportation of palms tree materials where R. ecuadoriensis are living.

Genetic data support speciation between Panstrongylus howardi and Panstrongylus chinai, vectors of Chagas disease in Ecuador.

Limited genetic data are currently available for three vectors of Chagas disease in Ecuador, Panstrongylus howardi, P. chinai, and P. rufotuberculatus. Previously regarded as mainly sylvatic, these species have been poorly studied. Recently, they have been more frequently reported in domiciles and peridomiciles and are now considered true secondary vectors of Chagas disease in a country where an estimated 200,000 people are infected by Trypanosoma cruzi, a causative agent of this disease. In order to fill this gap, we obtained DNA for sequencing from 53 insects belonging to these three species and mainly sampled from the two Ecuadorian provinces of Loja and Manabí. We used six mitochondrial loci (COI, COII, ND4, CytB, 16S, and 12S) and two nuclear ones (ITS2 and 18S). We interpreted the phylogenetic trees built with single and concatenated data through maximum likelihood, Bayesian Markov chain Monte Carlo, and maximum parsimony methods. We provide evidence that P. chinai and P. howardi are indeed two supported species closely related and derived from a common ancestor. Additionally, the phylogenetic position of P. rufotuberculatus was confirmed as being distant from P. chinai and P. howardi and clustered with Triatoma dimidiata, a species belonging to the Northern American Triatoma clade.

Molecular characterization of Rhodnius robustus specimens, potential vectors for Chagas disease in French Guiana, South America.

Insects of the genus Rhodnius are broadly involved in Chagas disease transmission. In French Guiana, where the disease remains a public health problem, R. robustus and R. pictipes are vectors, but so far few genetic analyses of these local species have been reported. Here, we explored three mitochondrial genes (Cytb, Lsu-rRNA, and ND1) and one nuclear gene (D2) in 49 adult specimens morphologically characterized as R. robustus. We analyzed genetic polymorphisms and haplotype distributions, and we built phylogenetic trees using the available GenBank sequences from R. robustus and related species. The molecular taxonomy analysis confirmed that 35 insects, closely related to Brazilian species and separated by a few mutations, truly belong to R. robustus; two others were attributed to the R. prolixus complex and for 12 no sequence was obtained. The geographical haplotype distribution indicates a likely geographical structuring and evidenced true differentiation between the two main urban centers, Cayenne and Saint-Laurent-du-Maroni.

Wild populations of Triatoma infestans: Compilation of positive sites and comparison of their ecological niche with domestic population niche.

BACKGROUND: For several years, the wild populations of Triatoma infestans, main vector of Trypanosoma cruzi causing Chagas disease, have been considered or suspected of being a source of reinfestation of villages. The number of sites reported for the presence of wild T. infestans, often close to human habitats, has greatly increased, but these data are scattered in several publications, and others obtained by our team in Bolivia have not been published yet. METHODOLOGY/PRINCIPAL FINDINGS: Herein is compiled the largest number of wild sites explored for the presence of T. infestans collected with two methods The standardized methods aimed to determine the relationship between wild T. infestans and the ecoregion, and the directed method help to confirm the presence/absence of triatomines in the ecoregions. Entomological indices were compared between ecoregions and an environmental niche modelling approach, based on bioclimatic variables, was applied. The active search for wild T. infestans in Bolivia suggests a discontinuous distribution from the Andean valleys to the lowlands (Chaco), while the models used suggest a continuous distribution between the two regions and very large areas where wild populations remain to be discovered. The results compile the description of different habitats where these populations were found, and we demonstrate that the environmental niches of wild and domestic populations, defined by climatic variables, are similar but not equivalent, showing that during domestication, T. infestans has conquered new spaces with wider ranges of temperature and precipitation. CONCLUSIONS/SIGNIFICANCE: The great diversity of wild T. infestans habitats and the comparison of their ecological niches with that of domestic populations confirm the behavioural plasticity of the species that increase the possibility of contact with humans. The result of the geographical distribution model of the wild populations calls for more entomological vigilance in the corresponding areas in the Southern Cone countries and in Bolivia. The current presentation is the most comprehensive inventory of wild T. infestans-positive sites that can be used as a reference for further entomological vigilance in inhabited areas.

Trypanosoma musculi Infection in Mice Critically Relies on Mannose Receptor-Mediated Arginase Induction by a TbKHC1 Kinesin H Chain Homolog.

Arginase activity induction in macrophages is an escape mechanism developed by parasites to cope with the host's immune defense and benefit from increased host-derived growth factor production. We report that arginase expression and activity were induced in macrophages during mouse infection by Trypanosoma musculi, a natural parasite of this host. This induction was reproduced in vitro by excreted/secreted factors of the parasite. A mAb directed to TbKHC1, an orphan kinesin H chain from Trypanosoma brucei, inhibited T. musculi excreted/secreted factor-mediated arginase induction. Anti-TbKHC1 Ab also inhibited T. musculi growth, both in vitro and in vivo. Induction of arginase activity and parasite growth involved C-type lectin receptors, because mannose injection decreased arginase activity induction and parasite load in vitro and in vivo. Accordingly, the parasite load was reduced in mice lacking mannose receptor C-type 1. The T. musculi KHC1 homolog showed high similarity with TbKHC1. Bioinformatics analysis revealed the presence of homologs of this gene in other trypanosomes, including pathogens for humans and animals. Host metabolism dysregulation represents an effective parasite mechanism to hamper the host immune response and modify host molecule production to favor parasite invasion and growth. Thus, this orphan kinesin plays an important role in promoting trypanosome infection, and its neutralization or the lock of its partner host molecules offers promising approaches to increasing resistance to infection and new developments in vaccination against trypanosomiasis.

Molecular identification of wild triatomines of the genus Rhodnius in the Bolivian Amazon: Strategy and current difficulties.

The Amazon region has recently been considered as endemic in Latin America. In Bolivia, the vast Amazon region is undergoing considerable human migrations and substantial anthropization of the environment, potentially renewing the danger of establishing the transmission of Chagas disease. The cases of human oral contamination occurring in 2010 in the town of Guayaramerín provided reasons to intensify research. As a result, the goal of this study was to characterize the species of sylvatic triatomines circulating in the surroundings of Yucumo (Beni, Bolivia), a small Amazonian city at the foot of the Andes between the capital (La Paz) and Trinidad the largest city of Beni. The triatomine captures were performed with mice-baited adhesive traps mostly settled in palm trees in forest fragments and pastures. Species were identified by morphological observation, dissection of genitalia, and sequencing of three mitochondrial gene fragments and one nuclear fragment. Molecular analysis was based on (i) the identity score of the haplotypes with GenBank sequences through the BLAST algorithm and (ii) construction of phylogenetic trees. Thirty-four triatomines, all belonging to the Rhodnius genus, of which two were adult males, were captured in palm trees in forest fragments and pastures (overall infestation rate, 12.3%). The morphology of the phallic structures in the two males confirmed the R. stali species. For the other specimens, after molecular sequencing, only one specimen was identified with confidence as belonging to Rhodnius robustus, the others belonged to one of the species of the Rhodnius pictipes complex, probably Rhodnius stali. The two species, R. robustus and R. stali, had previously been reported in the Alto Beni region (edge of the Amazon region), but not yet in the Beni department situated in the Amazon region. Furthermore, the difficulties of molecular characterization of closely related species within the three complexes of the genus Rhodnius are highlighted and discussed.

Over Six Thousand Trypanosoma cruzi Strains Classified into Discrete Typing Units (DTUs): Attempt at an Inventory.

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.

Blood meal sources of wild and domestic Triatoma infestans (Hemiptera: Reduviidae) in Bolivia: connectivity between cycles of transmission of Trypanosoma cruzi.

BACKGROUND: Chagas disease is a major public health problem in Latin America. Its etiologic agent, Trypanosoma cruzi, is mainly transmitted through the contaminated faeces of blood-sucking insects called triatomines. Triatoma infestans is the main vector in various countries in South America and recently, several foci of wild populations of this species have been described in Bolivia and other countries. These wild populations are suspected of affecting the success of insecticide control campaigns being carried out in South America. To assess the risk that these T. infestans populations pose to human health, it is helpful to determine blood meal sources. METHODS: In the present work, blood meals were identified in various Bolivian wild T. infestans populations and in three specific areas, in both wild and intra-peridomestic populations to assess the links between wild and domestic cycles of T. cruzi transmission. PCR-HDA and sequencing of Cytb gene were used to identify these blood meal sources. RESULTS AND DISCUSSION: Fourteen vertebrate species were identified as wild blood meal sources. Of those, the most prevalent species were two Andean endemic rodents, Octodontomys gliroides (36%) and Galea musteloides (30%), while humans were the third most prevalent source (18.7%). Of 163 blood meals from peridomestic areas, more than half were chickens, and the others were generally domestic animals or humans. Interestingly, blood from wild animals was identified in triatomines captured in the peridomestic and domestic environment, and blood from domestic animals was found in triatomines captured in the wild, revealing links between wild and domestic cycles of T. cruzi transmission. CONCLUSION: The current study suggests that wild T. infestans attack humans in the wild, but is also able to bite humans in domestic settings before going back to its natural environment. These results support the risk to human health posed by wild populations of T. infestans.

Reconsideration of the seven discrete typing units within the species Trypanosoma cruzi, a new proposal of three reliable mitochondrial clades.

It is generally acknowledged that Trypanosoma cruzi, responsible for Chagas disease, is structured into six or seven distinct discrete typing units (DTUs), and termed TcI through TcVI and TcBat for the seventh, by a collective of researchers. However, such structuring can be validated only when the species is analyzed over its entire distribution area with the same genetic markers. Many works have dealt with several DTUs in limited areas, generally one country, others have dealt with only one DTU over the endemic area, but no work has reported data of all DTUs over the entire endemic area. Hence, the aim of this minireview was to analyze three gene sequences, already deposited in GenBank by others, over the entire geographical distribution of Chagas disease. Two mitochondrial (CytB and COII) and one nuclear gene (Gpi) were selected (i) among those most widely used in the field, (ii) of single copy for the nuclear one, and (iii) presenting common sequences of sufficient size for applying phylogenetic tools. They were analyzed using maximum likelihood trees and phylogenetic networks. Remarkably, only three significant clusters instead of seven were found with the mitochondrial genes. With the nuclear gene, surprisingly, all seven expected clusters did not have significant bootstrap values. Moreover, DTUs TcV and TcVI were indistinguishable as were TcIII and TcIV. Additionally, we have undertaken a minireview of seventy-five publications presenting phylogenetic trees with identifiable DTUs that allowed us, together with our own results, to seriously question the structuring of T. cruzi into six or seven separated DTUs. We propose that mitochondrial typing in three clusters currently named mtTcI, mtTcII, and mtTcIII is robust whereas nuclear typing may lead to a questionable clustering but it is valuable for detecting mitochondrial introgression, heterozygous states and allelic composition.

Optimized multilocus sequence typing (MLST) scheme for Trypanosoma cruzi.

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.

Discriminating micropathogen lineages and their reticulate evolution through graph theory-based network analysis: the case of Trypanosoma cruzi, the agent of Chagas disease.

Micropathogens (viruses, bacteria, fungi, parasitic protozoa) share a common trait, which is partial clonality, with wide variance in the respective influence of clonality and sexual recombination on the dynamics and evolution of taxa. The discrimination of distinct lineages and the reconstruction of their phylogenetic history are key information to infer their biomedical properties. However, the phylogenetic picture is often clouded by occasional events of recombination across divergent lineages, limiting the relevance of classical phylogenetic analysis and dichotomic trees. We have applied a network analysis based on graph theory to illustrate the relationships among genotypes of Trypanosoma cruzi, the parasitic protozoan responsible for Chagas disease, to identify major lineages and to unravel their past history of divergence and possible recombination events. At the scale of T. cruzi subspecific diversity, graph theory-based networks applied to 22 isoenzyme loci (262 distinct Multi-Locus-Enzyme-Electrophoresis -MLEE) and 19 microsatellite loci (66 Multi-Locus-Genotypes -MLG) fully confirms the high clustering of genotypes into major lineages or "near-clades". The release of the dichotomic constraint associated with phylogenetic reconstruction usually applied to Multilocus data allows identifying putative hybrids and their parental lineages. Reticulate topology suggests a slightly different history for some of the main "near-clades", and a possibly more complex origin for the putative hybrids than hitherto proposed. Finally the sub-network of the near-clade T. cruzi I (28 MLG) shows a clustering subdivision into three differentiated lesser near-clades ("Russian doll pattern"), which confirms the hypothesis recently proposed by other investigators. The present study broadens and clarifies the hypotheses previously obtained from classical markers on the same sets of data, which demonstrates the added value of this approach. This underlines the potential of graph theory-based network analysis for describing the nature and relationships of major pathogens, thereby opening stimulating prospects to unravel the organization, dynamics and history of major micropathogen lineages.

Variations in time and space of an Andean wild population of T. infestans at a microgeographic scale.

BACKGROUND: Wild populations of Triatoma infestans are now believed to be the source of reinfestation of dwellings in some Andean areas and could impede the full achievement of vector control campaigns in this region. Given the poor knowledge of these populations in natural conditions, their basic biology traits, such as monthly demographic variations and movements of individuals, were explored. METHODS: A previously identified wild population of T. infestans in a field adjacent to a group of isolated houses in an Andean valley (department of La Paz, Bolivia) was explored using regular capture assays over 13 months in 50 sites selected at the beginning of the study. The capture-mark-recapture method was applied monthly using mouse-baited adhesive traps for captures and fingernail polish of different colors for the marking. RESULTS: The monthly capture assays did not show significant differences between rainy and dry seasons, showing evidence for a certain stability of the wild T. infestans population with only the nymph population tending to decline during the middle of the rainy season when rain is more intensive. Throughout the study, the monthly average number of bugs was 51.1 ± 25.3 per assay, 91.1% were nymphs, and they were found at 30 of the 50 sites (60%). The number of times a site was positive varied from one to 13. Site infestation was associated with the underground position of the traps, and rocks around and in the surroundings of the traps. The recaptures after marking were successful (138 recaptures over the study). The marking made it possible to detect for 14.5% of the recaptures significant movements of adults (up to 168 m) and nymphs (up to 34 m). Some bugs (nymphs and females) were recaptured after 5 months. For adults, recaptures (46 in total) mostly occurred between September and March. Females were recaptured twice as frequently as males. CONCLUSION: The Andean wild populations of T. infestans showed a strong spatial and temporal stability during the year-long study. Dispersal may occur mainly during the rainy season. The capture-mark-recapture method was successful and the longevity of the bugs and the distances covered by nymphs and adults were recorded.

Putative panmixia in restricted populations of Trypanosoma cruzi isolated from wild Triatoma infestans in Bolivia.

Trypanosoma cruzi, the causative agent of Chagas disease, is subdivided into six discrete typing units (DTUs; TcI-TcVI) of which TcI is ubiquitous and genetically highly variable. While clonality is the dominant mode of propagation, recombinant events play a significant evolutive role. Recently, foci of wild Triatoma infestans have been described in Bolivia, mainly infected by TcI. Hence, for the first time, we evaluated the level of genetic exchange within TcI natural potentially panmictic populations (single DTU, host, area and sampling time). Seventy-nine TcI stocks from wild T. infestans, belonging to six populations were characterized at eight microsatellite loci. For each population, Hardy-Weinberg equilibrium (HWE), linkage disequilibrium (LD), and presence of repeated multilocus genotypes (MLG) were analyzed by using a total of seven statistics, to test the null hypothesis of panmixia (H0). For three populations, none of the seven statistics allowed to rejecting H0; for another one the low size did not allow us to conclude, and for the two others the tests have given contradictory results. Interestingly, apparent panmixia was only observed in very restricted areas, and was not observed when grouping populations distant of only two kilometers or more. Nevertheless it is worth stressing that for the statistic tests of "HWE", in order to minimize the type I error (i. e. incorrect rejection of a true H0), we used the Bonferroni correction (BC) known to considerably increase the type II error ( i. e. failure to reject a false H0). For the other tests (LD and MLG), we did not use BC and the risk of type II error in these cases was acceptable. Thus, these results should be considered as a good indicator of the existence of panmixia in wild environment but this must be confirmed on larger samples to reduce the risk of type II error.