Comparative analysis of metacyclogenesis and infection curves in different discrete typing units of Trypanosoma cruzi.

Chagas disease (CD), caused by the complex life cycle parasite Trypanosoma cruzi, is a global health concern and impacts millions globally. T. cruzi's genetic variability is categorized into discrete typing units (DTUs). Despite their widespread presence in the Americas, a comprehensive understanding of their impact on CD is lacking. This study aims to analyze life cycle traits across life cycle stages, unraveling DTU dynamics. Metacyclogenesis curves were generated, inducing nutritional stress in epimastigotes of five DTUs (TcI (MG), TcI (DA), TcII(Y), TcIII, TcIV, and TcVI), resulting in metacyclic trypomastigotes. Infection dynamics in Vero cells from various DTUs were evaluated, exploring factors like amastigotes per cell, cell-derived trypomastigotes, and infection percentage. Statistical analyses, including ANOVA tests, identified significant differences. Varying onset times for metacyclogenesis converged on the 7th day. TcI (MG) exhibited the highest metacyclogenesis potential. TcI (DA) stood out, infecting 80% of cells within 24 h. TcI demonstrated the highest potential in both metacyclogenesis and infection among the strains assessed. Intra-DTU diversity was evident among TcI strains, contributing to a comprehensive understanding of Trypanosoma cruzi dynamics and genetic diversity.

Detection and Genotyping of Trypanosoma cruzi Samples in Species of Genus Rhodnius from Different Environments in the Brazilian Amazon.

Background: In the Amazon region, several species of triatomines occur in the natural environments. Among them, species of the genus Rhodnius are a risk to human populations due to their high rates of infection with Trypanosoma cruzi. The aim of this study was to identify the T. cruzi genotypes in Rhodnius specimens and their relationship with sylvatic hosts from different environments in the Brazilian Amazon. Methods: A total of 492 triatomines were collected from the municipalities of Monte Negro, Rondônia state, and Humaitá, Amazonas state, 382 of them being nymphs and 110 adults. Genotyping of T. cruzi in six discrete typing units (DTUs) was performed using conventional multilocus PCR. The triatomines that were positive for T. cruzi and engorged with blood were also targeted for amplification of the cytochrome B (cytB) gene to identify bloodmeal sources. Results: Of the 162 positive samples, the identified DTUs were TcI (87.65%) and TcIV (12.35%). It was observed that 102 specimens were engorged with a variety of bloodmeals. Triatomines infected with TcI were associated with DNA of all identified vertebrates, except Plecturocebus brunneus. TcIV was detected in triatomines that fed on Coendou prehensilis, Didelphis marsupialis, Mabuya nigropunctata, P. brunneus, Pithecia irrorata, Sapajus apella, and Tamandua tetradactyla. Conclusion: Results highlight the need to understand the patterns of T. cruzi genotypes in Rhodnius spp. and their association with sylvatic hosts to better elucidate their role in the transmission of Chagas disease in the Amazon region.

Fifteen Years after the Definition of Trypanosoma cruzi DTUs: What Have We Learned?

Trypanosoma cruzi, the protozoan causative of Chagas disease (ChD), exhibits striking genetic and phenotypic intraspecific diversity, along with ecoepidemiological complexity. Human-pathogen interactions lead to distinct clinical presentations of ChD. In 2009, an international consensus classified T. cruzi strains into six discrete typing units (DTUs), TcI to TcVI, later including TcBat, and proposed reproducible genotyping schemes for DTU identification. This article aims to review the impact of classifying T. cruzi strains into DTUs on our understanding of biological, ecoepidemiological, and pathogenic aspects of T. cruzi. We will explore the likely origin of DTUs and the intrinsic characteristics of each group of strains concerning genome organization, genomics, and susceptibility to drugs used in ChD treatment. We will also provide an overview of the association of DTUs with mammalian reservoirs, and summarize the geographic distribution, and the clinical implications, of prevalent specific DTUs in ChD patients. Throughout this review, we will emphasize the crucial roles of both parasite and human genetics in defining ChD pathogenesis and chemotherapy outcome.

Phylogenetic description of Trypanosoma cruzi isolates from Dipetalogaster maxima: Occurrence of TcI, TcIV, and TcIV-USA.

Trypanosoma cruzi is the parasite responsible for Chagas disease. The parasite has been classified into six taxonomic assemblages: TcI-TcVI and TcBat (aka Discrete Typing Units or Near-Clades). No studies have focused on describing the genetic diversity of T. cruzi in the northwestern region of Mexico. Within the Baja California peninsula lives Dipetalogaster maxima, the largest vector species for CD. The study aimed to describe the genetic diversity of T. cruzi within D. maxima. A total of three Discrete Typing Units (DTUs) were found (TcI, TcIV, and TcIV-USA). TcI was the predominant DTU found (∼75% of samples), in concordance with studies from the southern USA, one sample was described as TcIV while the other ∼20% pertained to TcIV-USA, which has recently been proposed to have enough genetic divergence from TcIV, to merit its own DTU. Potential phenotype differences between TcIV and TcIV-USA should be assessed in future studies.

Detection of Trypanosoma cruzi DTUs TcI and TcIV in two outbreaks of orally-transmitted Chagas disease in the Northern region of Brazil

ABSTRACT This study describes the laboratory investigation of two acute Chagas disease outbreaks that occurred in the riverside communities of Marimarituba and Cachoeira do Arua, in the Santarem municipality, Para State, located in the Northern region of Brazil, and occurred in March 2016 and August 2017, respectively. The generation of data regarding the diversity of Trypanosoma cruzi parasites circulating in the Amazon region is key for understanding the emergence and expansion of Chagas disease. This study aimed to identify T. cruzi Discrete Typing Units (DTUs) involved in two outbreaks of acute Chagas disease (ACD) directly from the patient's biological sample. Nested and multiplex PCR targeting the 24Sα (rRNA) and mini-exon genes, respectively, were used to identify T. cruzi DTU in blood samples from patients diagnosed with ACD. The samples with positive cPCR were submitted for analysis for T. cruzi DTUs, which included 13 samples from the patients with ACD by oral transmission and two samples collected from two newborns of two women with ACD, from Marimarituba and Cachoeira do Arua. The samples were classified as T. cruzi TcIV, from Marimarituba's outbreak, and T. cruzi TcI, from Cachoeira do Arua's outbreak. The molecular identification of T. cruzi may increase understanding of the role of this parasite in Chagas disease's emergence within the Amazon region, contributing to the improvement of the management of this important, but also neglected, disease.

Molecular characterization of Trypanosoma cruzi DTUs of the triatomine species in a Chagas disease endemic area.

Trypanosoma cruzi is the etiological agent of Chagas disease, a neglected tropical infection with great public health importance. This protozoan has triatomine insects as vector but may also be transmitted through blood transfusion, organ transplants, ingestion of contaminated food, or congenitally. It has a heterogeneous population classified into Discrete Typing Units (DTUs), TcI-TcVI and TcBat. The aim of this study was to molecularly characterize the DTUs of T. cruzi in triatomines from a Chagas disease endemic area in Northeastern Brazil. Triatomines were collected and the gut content was microscopically analyzed to investigate the presence of trypanosomatid flagellates. In addition, digestive tracts of some specimens were dissected and molecularly analyzed through PCR for Trypanosoma spp. and sequencing. PCR positive samples were further submitted to a multiplex PCR for DTUs of T. cruzi. A total of 117 triatomines were collected, 93.16% being in intradomicile and 6.84% in peridomicile environments. Insects were identified as Panstrongylus lutzi (37.60%), Triatoma pseudomaculata (26.50%), Triatoma brasiliensis (23.08%) and Panstrongylus megistus (12.82%). The specimens herein analyzed presented infection rates by T. cruzi of 5.49% and 12.09% in parasitological and molecular examinations, respectively. Multiplex PCR screening revealed 70.59% of the TcI genotype, detected in all triatomine species identified in this study and 29.41% of the DTU TcIII/TcIV detected in P. megistus and P. lutzi. T. cruzi infect triatomines in intradomicile and peridomicile environments, which brings attention to the risk of human infections and to the importance of the implementation of surveillance and entomological control actions.

Genetic Diversity of Trypanosoma cruzi in Panama Inferred by Multi-locus Sequence Typing of Mitochondrial Genes.

The objective of this study was to provide information on Trypanosoma cruzi genetic diversity among isolates obtained from different biological sources circulating in endemic areas of Panama. Initial discrete typing units (DTUs) assignment was performed evaluating three single locus molecular markers (mini-exon, heat shock protein 60 and glucose-6-phosphate isomerase genes). Further diversity within TcI lineages was explored using a multi-locus sequence typing approach with six maxicircle genes. Haplotype network analysis and evolutionary divergency estimations were conducted to investigate the genetic relatedness between Panamanian TcI isolates and isolates from different endemic regions in the Americas. Our molecular approach validated that TcI is the predominant DTU circulating in Panama across different hosts and vector species, but also confirmed the presence of TcIII and TcVI circulating in the country. The phylogenetic tree topography for most Panamanian TcI isolates displayed a high level of genetic homogeneity between them. The haplotype network analysis inferred a higher genetic diversity within Panamanian TcI isolates, displaying eight different haplotypes circulating in endemic regions of the country, and revealed geographical structuring among TcI from different endemic regions in the Americas. This study adds novelty on the genetic diversity of T. cruzi circulating in Panama and complements regional phylogeographic studies regarding intra-TcI variations.

Trypanosoma cruzi genetic diversity: impact on transmission cycles and Chagas disease

Trypanosoma cruzi, the agent of Chagas disease (ChD), exhibits remarkable biological and genetic diversity, along with eco-epidemiological complexity. In order to facilitate communication among researchers aiming at the characterisation of biological and epidemiological aspects of T. cruzi, parasite isolates and strains were partitioned into seven discrete typing units (DTUs), TcI-TcVI and TcBat, identifiable by reproducible genotyping protocols. Here we present the potential origin of the genetic diversity of T. cruzi and summarise knowledge about eco-epidemiological associations of DTUs with mammalian reservoirs and vectors. Circumstantial evidence of a connection between T. cruzi genotype and ChD manifestations is also discussed emphasising the role of the host's immune response in clinical ChD progression. We describe genomic aspects of DTUs focusing on polymorphisms in multigene families encoding surface antigens that play essential functions for parasite survival both in the insect vector and the mammalian host. Such antigens most probably contributed to the parasite success in establishing infections in different hosts and exploring several niches. Gaps in the current knowledge and challenges for future research are pointed out.

Shelter cats host infections with multiple Trypanosoma cruzi discrete typing units in southern Louisiana.

Trypanosoma cruzi is a zoonotic parasite endemic in the southern US and the Americas, which may frequently infect dogs, but limited information is available about infections in cats. We surveyed a convenience sample of 284 shelter cats from Southern Louisiana to evaluate T. cruzi infection using serological and PCR tests. Parasites from PCR positive cats were also genotyped by PCR and deep sequencing to assess their genetic diversity. We detected a seropositivity rate for T. cruzi of at least 7.3% (17/234), and 24.6% of cats (70/284) were PCR positive for the parasite. Seropositivity increased with cat age (R2 = 0.91, P = 0.011), corresponding to an incidence of 7.2% ± 1.3 per year, while PCR positivity decreased with age (R2 = 0.93, P = 0.007). Cats were predominantly infected with parasites from TcI and TcVI DTUs, and to a lesser extent from TcIV and TcV DTUs, in agreement with the circulation of these parasite DTUs in local transmission cycles. These results indicate that veterinarians should have a greater awareness of T. cruzi infection in pets and that it would be important to better evaluate the risk for spillover infections in humans.

Serological Approaches for Trypanosoma cruzi Strain Typing.

Trypanosoma cruzi, the protozoan agent of Chagas' disease, displays a complex population structure made up of multiple strains showing a diverse ecoepidemiological distribution. Parasite genetic variability may be associated with disease outcome, hence stressing the need to develop methods for T. cruzi typing in vivo. Serological typing methods that exploit the presence of host antibodies raised against polymorphic parasite antigens emerge as an appealing approach to address this issue. These techniques are robust, simple, cost-effective, and are not curtailed by methodological/biological limitations intrinsic to available genotyping methods. Here, we critically assess the progress towards T. cruzi serotyping and discuss the opportunity provided by high-throughput immunomics to improve this field.

Differential tissue distribution of discrete typing units after drug combination therapy in experimental Trypanosoma cruzi mixed infection.

The aim of the present work was to evaluate the distribution of the different clones of the parasite prevailing after treatment with benznidazole (BZ) and clomipramine (CLO), in mice infected with Trypanosoma cruzi, Casibla isolate which consists of a mixture of two discrete typing units (DTUs). Albino Swiss mice were infected and treated with high and low concentrations of BZ (100 or 6.25 mg/kg), CLO (5 or 1.25 mg/kg), or the combination of both low doses (BZ6.25 + CLO1.25), during the acute phase of experimental infection. Treatment efficacy was evaluated by comparing parasitaemia, survival and tissular parasite presence. For DTUs genotyping, blood, skeletal and cardiac muscle samples were analysed by multiplex quantitative polymerase chain reaction. The combined treatment had similar outcomes to BZ6.25; BZ100 was the most effective treatment, but it failed to reach parasite clearance and produced greater histological alterations. Non-treated mice and the ones treated with monotherapies showed both DTUs while BZ6.25 + CLO1.25 treated mice showed only TcVI parasites in all the tissues studied. These findings suggest that the treatment may modify the distribution of infecting DTUs in host tissues. Coinfection with T. cruzi clones belonging to different DTUs reveals a complex scenario for the treatment of Chagas disease and search for new therapies.

Natural infection with Trypanosoma cruzi in three species of non-human primates in southeastern Mexico: A contribution to reservoir knowledge.

The mechanisms of infection and dispersion of Trypanosoma cruzi among animals, especially in the sylvatic environment, are still not entirely clear, and various aspects of the transmission dynamics of this parasite in the sylvatic environment are still unknown. T. cruzi is a parasite with a great biological and genetic diversity that infects a wide variety of hosts, therefore, transmission cycles of this parasite are complex. The objective of this study was to determine the prevalence of T. cruzi infection and analyze the genetic variability of the discrete typing units (DTUs) of the parasite in three non-human primate species (Alouatta palliata, Alouatta pigra, and Ateles geoffroyi) in southeastern Mexico. A total of one hundred sixty-four serum samples (42 samples of A. pigra, 41 samples of A. palliata (free-ranging) and 81 samples of A. geoffroyi (hosted in care centers)) were analyzed for the detection of anti-T. cruzi antibodies by ELISA assays. The seroprevalence of infection was 23.39% in A. palliata, 21.40% in A. pigra and 16.27% in A. geoffroyi. Additionally, presence of parasite DNA was assessed by PCR, and the identification of DTUs was performed by real-time PCR coupled to High Resolution Melting (qPCR-HRM). Different DTUs (TcI, TcII, TcIII, TcV and TcVI) were found in the analyzed monkeys. In addition, infection of monkeys was not associated with age or gender, but it was associated with the species. This study reveals the risk of infection in the study area and that the different DTUs of the parasite can coexist in the same habitat, indicating that T. cruzi transmission in the study area is very complex and involves many ecological factors. However, there is a need for long-term studies of host-parasite interactions to provide a solid understanding of the ecology of these species and to understand the dispersion strategies of T. cruzi.

Organs infected with Trypanosoma cruzi and DTU identification in the naturally infected rodent Octodon degus.

Chagas disease is a public health problem in America. Its parasite, Trypanosoma cruzi, presents different discrete typing units (DTUs), colonizes organs of mammalian hosts in chronic infections, and presents tropism for particular organs in experimental infections. We evaluated T. cruzi tropism towards organs on the naturally infected rodent Octodon degus, identifying the parasites' DTUs, by means of conventional PCR and hybridization. Almost all the analyzed organs presented T. cruzi. More than 42% of the tested oesophagus, skin, skeletal muscle, brain and intestine showed T. cruzi DNA. Other nine types of organs were infected in over 15%. These results suggest that there is some tropism by T. cruzi in chronically infected O. degus. DTU TcV was present in 92.5% of infected organs with identified DTUs; this DTU is frequently reported in human infections in the Southern Cone of South America. Few organs showed mixed DTU infections. This is one of the few reports on the outcome of chronic natural T. cruzi-infection in wild mammal hosts exposed to naturally infected vectors.

Reinfestation with Triatoma infestans despite vigilance efforts in the municipality of Saipina, Santa Cruz, Bolivia: Situational description two months after fumigation.

Chagas disease is still a major public health problem in Bolivia mostly due to the recurrent reinfestation of houses by Triatoma infestans. The current study evaluated the danger of reinfesting bugs by determining their infection rate, the genetic group (discrete typing unit, DTU) of Trypanosoma cruzi that infect them, and the possible association of recurrent infestation with environmental variables. In the municipality of Saipina, 254 km from Santa Cruz de la Sierra, 57 dwellings with reinfestation background and the latest fumigation 1 or 2 months before were actively searched for triatomines. The infection of the bugs and the DTUs of T. cruzi were determined with PCR methods. Microenvironmental variables were estimated surfaces of the different ground covers around each dwelling. Principal component analysis (PCA) and logistic regression were applied to the data set. Among the houses visited, 54.4% were still infested with T. infestans, and 201 T. infestans were captured, 56% indoors and 43.8% outdoors. The infection rate with T. cruzi was 24%. The TcII/TcV/TcVI group of DTUs was 80%, while TcI and TcIII/TcIV had equal values of 10%. No significant differences of DTU distribution were found between nymphs and adults, females and males, nor between intradomicile and peridomicile areas. PCA identified urban and nonurban dwellings: the former was associated with intradomicile reinfestation by nymphs. From the logistic regression analyses, the intradomicile reinfestation tended to be associated with the peridomicile around dwellings. In contrast, peridomicile infestation was more associated with sylvatic areas. Interestingly, the presence of fields (pasture, crops) around the dwelling might have a protective role regarding reinfestation. The results show that vector control actions fail, and the inhabitants of the municipality of Saipina continue to be exposed to T. cruzi transmission risk.

Co-infection with distinct Trypanosoma cruzi strains induces an activated immune response in human monocytes.

AIMS: The aim of the study was to evaluate the immune response triggered by the first contact of human monocytes with two T cruzi strains from distinct discrete typing units (DTUs) IV and V, and whether co-infection with these strains leads to changes in monocyte immune profiles, which could in turn influence the subsequent infection outcome. METHODS AND RESULTS: We evaluated the influence of in vitro single- and co-infection with AM64 and 3253 strains on immunological characteristics of human monocytes. Single infection of monocytes with AM64 or 3253 induced opposing anti-inflammatory and inflammatory responses, respectively. Co-infection was observed in over 50% of monocytes after 15 hours of culture, but this percentage dropped ten-fold after 72 hours. Co-infection led to high monocyte activation and an increased percentage of both IL-10 and TNF. The decreased percentage of co-infected cells observed after 72 hours was associated with a decreased frequency of TNF-expressing cells. CONCLUSION: Our results show that the exacerbated response observed in co-infection with immune-polarizing strains is associated with a decreased frequency of co-infected cells, suggesting that the activated response favours parasite control. These findings may have implications for designing new Chagas disease preventive strategies.

Genotyping of Trypanosoma cruzi from Clinical Samples by Multilocus Conventional PCR.

By the most recent nomenclature, Trypanosoma cruzi isolates are classified into six discrete typing units (DTUs)-T. cruzi I to T. cruzi VI and TcBat. One of the major challenges in the Chagas disease study is to find an association between DTUs and clinical manifestations of the disease or response to treatment. Herein, a protocol based on the amplification of T. cruzi SL-IRac, SL-IR I and II, 24Sα rDNA, and A10 targets by multilocus conventional PCRs is described. Following this methodology, it is possible to perform the genotyping directly from the blood and other clinical samples, without the need to isolate the parasite prior to the DNA extraction, even in a lower parasite concentration. Furthermore, this methodology increases the probability to detect mixed infections, avoiding a possible selection of strains during the parasite isolation.

Differential tissue distribution of Trypanosoma cruzi during acute experimental infection: Further evidence using natural isolates.

In the present work, we evaluated the effect of mixed Trypanosoma cruzi infections, studying the biological distribution of the different parasites in blood, heart and skeletal muscle during the acute phase. Albino Swiss mice were infected with different parasite strain/isolates or with a combination of them. The parasites in the different tissues were typified through specific PCR, population variability was analyzed through RFLP studies and parasitological and histopathological parameters were evaluated. We found a predominance of TcII and TcVI in all tissues samples respect to TcV and different parasite populations were found in circulation and in the tissues from the same host. These results verify the distribution of parasites in host tissues from early stages of infection and show biological interactions among different genotypes and populations of T. cruzi.

Molecular diagnosis of Trypanosoma cruzi.

Chagas disease, caused by the kinetoplastid protozoan Trypanosoma cruzi, affects millions of people, most of them neglected populations. The different phases of the disease, the transmission mode and the high genetic variability of the parasite determine that molecular detection methods display different degree of success. Molecular diagnostic tests may be employed during epidemiological surveys of transmission, for early diagnosis of congenital transmission and acute infections due to oral transmission, transfusion or transplantation routes, reactivation due to immunosuppression and monitoring of treatment response in chronically infected patients receiving trypanocidal chemotherapy. This manuscript summarizes the most widely used molecular tools to detect T. cruzi infection in different epidemiological and clinical scenarios.

Trypanosoma cruzi genetic diversity: Something new for something known about Chagas disease manifestations, serodiagnosis and drug sensitivity.

The genetic diversity of Trypanosoma cruzi, the protozoan agent of Chagas disease, is widely recognized. At present, T. cruzi is partitioned into seven discrete typing units (DTUs), TcI-TcVI and Tcbat. This article reviews the present knowledge on the parasite population structure, the evolutionary relationships among DTUs and their distinct, but not exclusive ecological and epidemiological associations. Different models for the origin of hybrid DTUs are examined, which agree that genetic exchange among T. cruzi populations is frequent and has contributed to the present parasite population structure. The geographic distribution of the prevalent DTUs in humans from the southern United States to Argentina is here presented and the circumstantial evidence of a possible association between T. cruzi genotype and Chagas disease manifestations is discussed. The available information suggests that parasite strains detected in patients, regardless of the clinical presentation, reflect the principal DTU circulating in the domestic transmission cycles of a particular region. In contrast, in several orally transmitted outbreaks, sylvatic strains are implicated. As a consequence of the genotypic and phenotypic differences of T. cruzi strains and the differential geographic distribution of DTUs in humans, regional variations in the sensitivity of the serological tests are verified. The natural resistance to benznidazole and nifurtimox, verified in vivo and in vitro for some parasite stocks, is not associated with any particular DTU, and does not explain the marked difference in the anti-parasitic efficacy of both drugs in the acute and chronic phases of Chagas disease. Throughout this review, it is emphasized that the interplay between parasite and host genetics should have an important role in the definition of Chagas disease pathogenesis, anti-T. cruzi immune response and chemotherapy outcome and should be considered in future investigations.

Inactive trans-Sialidase Expression in iTS-null Trypanosoma cruzi Generates Virulent Trypomastigotes.

Disclosing virulence factors from pathogens is required to better understand the pathogenic mechanisms involved in their interaction with the host. In the case of Trypanosoma cruzi several molecules are associated with virulence. Among them, the trans-sialidase (TS) has arisen as one of particular relevance due to its effect on the immune system and involvement in the interaction/invasion of the host cells. The presence of conserved genes encoding for an inactive TS (iTS) isoform is puzzlingly restricted to the genome of parasites from the Discrete Typing Units TcII, TcV, and TcVI, which include highly virulent strains. Previous in vitro results using recombinant iTS support that this isoform could play a different or complementary pathogenic role to that of the enzymatically active protein. However, direct evidence involving iTS in in vivo pathogenesis and invasion is still lacking. Here we faced this challenge by transfecting iTS-null parasites with a recombinant gene that allowed us to follow its expression and association with pathological events. We found that iTS expression improves parasite invasion of host cells and increases their in vivo virulence for mice as shown by histopathologic findings in heart and skeletal muscle.