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ÍtemSolo MetadatosUnderstanding the role of dogs (Canis lupus familiaris) in the transmission dynamics of trypanosoma cruzi genotypes in Colombia(2013-09-01) Ramírez, Juan David; Turriago, Brenda; Tapia-Calle, Gabriela; Guhl, FelipeThe dog (Canis lupus familiaris) is the most important domestic reservoir of Chagas disease, a zoonosis that affects more than 10 million people in Latin America. Trypanosoma cruzi, the etiologic agent of the disease, displays remarkable genetic variability, as indicated by its six genotypes (TcI-TcVI). A pilot study was conducted to establish the prevalence of T. cruzi among the canine population by analyzing 80 dogs. We report the identification of the TcI, TcII, TcIV and TcVI genotypes as single infections. TcI/TcII and TcI/TcIV presented as mixed infections and included the presence of Trypanosoma angel. The implications of this distribution are herein discussed. Based on the molecular epidemiology findings, this study suggests a plausible role for canine synanthropism in the transmission of T. cruzi.- ÍtemSolo MetadatosRetrospective molecular integrated epidemiology of chagas disease in Colombia(2013-12) Guhl, Felipe; Ramírez, Juan DavidAmerican trypanosomiasis is a very complex zoonosis that is present throughout South America, Central America, and Mexico and continues to represent a serious threat to the health of countries in the region. The parasite infects 150 species from 24 families of domestic and wild mammals and shows remarkable genetic variability evinced in at least seven discrete typing units (DTU’s) named TcI–TcVI with the presence of a novel genotype associated with bats named TcBat. These DTUs show a wide range of geographical and host distributions. Our study aimed to establish the relationship between the genetic diversity of Trypanosoma cruzi and the diverse clinical manifestations of infected patients and unravel the molecular eco-epidemiology in the epizootic and enzootic scenarios in Colombia. We undertook intensive sampling in 17 departments of Colombia among 11 triatomine species, 9 mammalian reservoir species and humans and obtained 637 biological clones that were subsequently analyzed using nuclear and mitochondrial molecular markers. TcI was the most prevalent (80.7%) followed by TcII (7.2%), TcIII (3.9%), TcIV (5%), TcV (0.8%), TcVI (1.6%) and TcBat (0.8%). Within domestic foci, TcI (70%) was the most prevalent, followed by TcII (20%), TcIII (1.6%), TcIV (3.6%), TcV (2.2%) and TcVI (2,6%); within sylvatic foci, TcI (85%) was the most prevalent, followed by TcII (0.3%), TcIII (5.5%), TcIV (7%), TcVI (1.1%) and TcBat (1.1%). The results suggest the occurrence of the seven DTUs and strict associations of independent DTUs with the host and environment in Colombia. The implications are discussed herein.
- ÍtemSolo MetadatosValidation of a poisson-distributed limiting dilution assay (LDA) for a rapid and accurate resolution of multiclonal infections in natural Trypanosoma cruzi populations(2013-02-15) Ramírez, Juan David; Herrera, Claudia; Bogotá, Yizeth; Duque, María Clara; Suárez-Rivillas, Alejandro; Guhl, FelipeTrypanosoma cruzi is the causative agent of American trypanosomiasis, a complex zoonotic disease that affects more than 10 million people in the Americas. Strains of this parasite possess a significant amount of genetic variability and hence can be divided into at least six discrete typing units (DTUs). The life cycle of this protist suggests that multiclonal infections may emerge due to the likelihood of contact of triatomine insects with more than 100 mammal species. To date, there have been a few studies on but no consensus regarding standardised methodologies to identify multiclonal infections caused by this parasite. Hence, the aim of this study was to develop and validate a limiting dilution assay (LDA) to identify multiclonal infections in T. cruzi populations by comparing the feasibility and reliability of this method with the widely applied solid phase blood agar (SPBA) methodology. We cloned reference strains belonging to three independent genotypes (TcI, TcII, and TcIV) and mixed infections (TcI + TcII) using LDA and SPBA; the comparison was conducted by calculating the feasibility and reliability of the methods employed. Additionally, we implemented LDA in strains recently isolated from Homo sapiens, Rhodnius prolixus, Triatoma venosa, Panstrongylus geniculatus, Tamandua tetradactyla, Rattus rattus, Didelphis marsupialis and Dasypus novemcinctus, with the aim of resolving multiclonal infections using molecular characterization employing SL-IR (spliced leader intergenic region of mini-exon gene), the 24S? rDNA gene and microsatellite loci. The results reported herein demonstrate that LDA is an optimal methodology to distinguish T. cruzi subpopulations based on microsatellite markers by showing the absence of multiple peaks within a single locus. Conversely, SPBA showed patterns of multiple peaks within a single locus suggesting multiclonal events. The biological consequences of these results and the debate between multiclonality and aneuploidy are discussed.
- ÍtemSolo MetadatosTrypanosome species in neo-tropical bats: biological, evolutionary and epidemiological implications(2014-03) Ramírez, Juan David; Tapia-Calle, Gabriela; Muñoz-Cruz, Geissler; Poveda, Cristina; Rendón, Lina M.; Hincapié, Eduwin; Guhl, FelipeBats (Chiroptera) are the only mammals naturally able to fly. Due to this characteristic they play a relevant ecological role in the niches they inhabit. These mammals spread infectious diseases from enzootic to domestic foci. Rabbies, SARS, fungi, ebola and trypanosomes are the most common pathogens these animals may host. We conducted intensive sampling of bats from the phyllostomidae, vespertilionidae and emballonuridae families in six localities from Casanare department in eastern Colombia. Blood-EDTA samples were obtained and subsequently submitted to analyses of mitochondrial and nuclear genetic markers in order to conduct barcoding analyses to discriminate trypanosome species. The findings according to the congruence of the three molecular markers suggest the occurrence of Trypanosoma cruzi cruzi (51%), T. c. marinkellei (9%), T. dionisii (13%), T. rangeli (21%), T. evansi (4%) and T. theileri (2%) among 107 positive bat specimens. Regarding the T. cruzi DTUs, we observed the presence of TcI (60%), TcII (15%), TcIII (7%), TcIV (7%) and TcBAT (11%) being the first evidence to our concern of the foreseen genotype TcBAT in Colombia. These results allowed us to propose reliable hypotheses regarding the ecology and biology of the bats circulating in the area including the enigmatic question whether TcBAT should be considered a novel DTU. The epidemiological and evolutionary implications of these findings are herein discussed.
- ÍtemSolo MetadatosFrom ancient to contemporary molecular eco-epidemiology of Chagas disease in the Americas(2014) Guhl, Felipe; Auderheide, Arthur; Ramírez, Juan DavidOne of the best-studied populations with regard to Chagas disease is from the coastal area of northern Chile at the foot of the western Andean slopes. The extremely arid climate here generates rapid, spontaneous desiccation of buried bodies, arresting the decay process. The absence of rainfall then preserves these dried bodies (mummies) for millennia. The aim of the present study was to perform the first molecular paleoepidemiological study on a set of 43 mummified human remains from the Atacama Desert in Northern Chile in order to elucidate the transmission dynamics and determinants of ancient genotypes, to try to unravel the natural history of the Trypanosoma cruzi taxon and Chagas disease. Interestingly, TcBat, a recently described Discrete Taxonomic Unit, emerges as the plausible ancestor of T. cruzi. The findings herein presented allow us to present a plausible model of T. cruzi transmission in pre-Columbian civilisations.
- ÍtemSolo MetadatosPrevalence of Trypanosoma cruzi's Discrete Typing Units in a cohort of Latin American migrants in Spain(2016) Martinez-Perez, Angela; Poveda, Cristina; Ramírez, Juan David; Norman, Francesca; Gironés, Núria; Guhl, Felipe; Monge-Maillo, Begoña; Fresno, Manuel; López-Vélez, RogelioChagas disease is caused by the protozoan Trypanosoma cruzi. This is an endemic disease in the Americas, but increased migration to Europe has made it emerge in countries where it was previously unknown, being Spain the second non endemic country in number of patients. T. cruzi is a parasite with a wide genetic diversity, which has been grouped by consensus into 6 Discrete Typing Units (DTUs) affecting humans. Some authors have linked these DTUs either to a specific epidemiological context or to the different clinical presentations. Our main objective was to describe the T. cruzi DTUs identified from a population of chronically infected Latin American migrants attending a reference clinic in Madrid. 149 patients meeting this condition were selected for the study. Molecular characterization was performed by an algorithm that combines PCR of the intergenic region of the mini exon-gene, the 24S? and 18S regions of rDNA and the variable region of the satellite DNA. A descriptive analysis was performed and associations between geographical/clinical data and the different DTUs were tested. DTUs could be determined in 105 out of 149 patients, 93.3% were from Bolivia, 67.7% were women and median age was 35 years (IQR 29-44). The most common DTU found was TcV (58; 55.2%), followed by TcIV (17; 16.2%), TcII (10; 9.5%) and TcI (4; 3.8%). TcIII and TcVI were not identified from any patient, and 15.2% patients presented mixed infections. In addition, we determined DTUs after treatment in a subset of patients. In 57% patients had different DTUs before and after treatment. DTUs distribution from this study indicates active transmission of T. cruzi is occurring in Bolivia, in both domestic and sylvatic cycles. TcIV was confirmed as a cause of chronic human disease. The current results indicate no correlation between DTU and any specific clinical presentation associated with Chagas disease, nor with geographical origin. Treatment with benznidazole does not always clear T. cruzi's genetic material from blood, and DTUs detected in the same patient may vary over time indicating that polyparasitism is frequent. © 2016 Elsevier B.V.
- ÍtemSolo MetadatosDistribution of Trypanosoma cruzi discrete typing units in bolivian migrants in Spain(2014-01) Perez-Molina, José A.; Poveda, Cristina; Martinez-Perez, Angela; Guhl, Felipe; Monge-Maillo, Begoña; Fresno, Manuel; López-Velez, Rogelio; Ramírez, Juan David; Girones, NuriaChagas disease is caused by the protozoan Trypanosoma cruzi. This parasite is transmitted to humans mainly through the faeces of infected triatomine “kissing” bugs, by blood transfusions or organ donation from infected donors, and can be transmitted from mother to child. This disease is endemic in the Americas, where Bolivia has up to 28.8% prevalence in general population. Increased migration to Europe has made it emerge in countries where it was previously unknown, being Spain the second country in number of patients after the United States. T. cruzi is an organism with a rich genetic diversity, what has been grouped into six discrete typing units (DTUs). Some authors have linked these DTUs either to specific geographical distribution or to the different clinical presentations. Nevertheless little is known about its distribution in migrant populations. Our aim was to describe the T. cruzi strains isolated from a population of chronically infected Bolivian patients attending our clinic in Madrid. Thirty-three consecutive patients meeting this condition were selected for the study. Molecular characterization was performed by an algorithm that combines PCR of the intergenic region of the mini exon-gene, the 24S? and 18S regions of rDNA and the variable region of the satellite DNA. A descriptive analysis was performed and associations between epidemiological/clinical data and the different DTUs were tested. Twenty-seven out of thirty-three patients had their DTU detected. Mean age was 36 years (IQR 31–43.3) and 23 were women (76.7%). The median time since arrival to Spain was 60 months (IQR 43–81). The most common DTU were TcV, TcIV and TcI. Four patients had cardiac involvement: 2 had TcV and 2 could not have their DTU determined. TcIII was not isolated from any patient. DTUs distribution in migrant population seems to be similar to that observed in the patients’ countries of origin.
- ÍtemSolo MetadatosContemporary cryptic sexuality in Trypanosoma cruzi(2012-07-09) Ramírez, Juan David; Guhl, Felipe; Messenger, Louisa A.; Lewis, Michael D.; Montilla, Marleny; Cucunuba, Zulma; Miles, Michael A.; Llewellyn, Martin S.Clonal propagation is considered to be the predominant mode of reproduction among many parasitic protozoa. However, this assumption may overlook unorthodox, infrequent or cryptic sexuality. Trypanosoma cruzi , which causes Chagas disease, is known to undergo non?Mendelian genetic exchange in the laboratory. In the field, evidence of extant genetic exchange is limited. In this study, we undertook intensive sampling of T. cruzi Discrete Typing Unit I in endemic eastern Colombia. Using Fluorescence?activated cell sorting, we generated 269 biological clones from 67 strains. Each clone was genotyped across 24 microsatellite loci. Subsequently, 100 representative clones were typed using 10 mitochondrial sequence targets (3.76?Kbp total). Clonal diversity among humans, reservoir hosts and vectors suggested complex patterns of superinfection and/or coinfection in oral and vector?borne Chagas disease cases. Clonal diversity between mother and foetus in a congenital case demonstrates that domestic TcI genotypes are infective in utero . Importantly, gross incongruence between nuclear and mitochondrial markers is strong evidence for widespread genetic exchange throughout the data set. Furthermore, a confirmed mosaic maxicircle sequence suggests intermolecular recombination between individuals as a further mechanism of genetic reassortment. Finally, robust dating based on mitochondrial DNA indicates that the emergence of a widespread domestic TcI clade that we now name TcIDOM (formerly TcIa/VENDom) occurred 23?000?±?12?000?years ago and was followed by population expansion, broadly corresponding with the earliest human migration into the Americas.
- ÍtemSolo MetadatosMultilocus PCR-RFLP profiling in Trypanosoma cruzi I highlights an intraspecific genetic variation pattern(2012-12) Ramírez, Juan David; Duque, María Clara; Montilla, Marleny; Cucunubá, Zulma M.; Guhl, FelipeChagas disease represents a serious problem in public health. This zoonotic pathology is caused by the kinetoplastid Trypanosoma cruzi which displays a high genetic diversity falling into six Discrete Typing Units (TcI–TcVI). In Colombia, the prevalent DTU is TcI with findings of TcII, TcIII and TcIV in low proportions. The aim of this work was to observe the genetic variability within TcI using a multilocus PCR-RFLP strategy. We analyzed 70 single-celled clones from triatomines, reservoirs and humans that were amplified and restricted via ten PCR-RFLPs targets across TcI genome, the restriction fragments were used to construct phylograms according to calculated genetic distances. We obtained five polymorphic targets (1f8, HSP60, HSP70, SAPA and H1) and the consensus tree constructed according to these regions allowed us to observe two well-defined groups with close association to the transmission cycles (domestic/peridomestic and sylvatic) of Chagas disease in Colombia. Our findings allowed us to corroborate the previous reported genotypes based on the intergenic region of mini-exon gene. More studies examining the genetic diversity among T. cruzi I populations must be conducted in order to obtain a better understanding in regions where this DTU is endemic.
