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2 Acta Tropica 119 (2011) 1 4 Contents lists available at ScienceDirect Acta Tropica journal homepage: Review Trypanosoma cruzi I diversity: Towards the need of genetic subdivision? Felipe Guhl, Juan David Ramírez Centro de Investigaciones en Microbiología y Parasitología Tropical, CIMPAT, Universidad de los Andes, Cra 1 No. 18A-10, Bogotá, Colombia article info abstract Article history: Received 23 February 2011 Received in revised form 28 March 2011 Accepted 1 April 2011 Available online 9 April 2011 Keywords: Trypanosoma cruzi I DTUs Genetic variability Mini-exon Microsatellites Trypanosoma cruzi the aethiological agent of Chagas disease, a complex zoonoses that affects the American continent is a genetically variable parasite subdivided into six Discrete Typing Units (DTUs). T. cruzi I is the most prevalent DTU affecting the northern countries of America with sporadical cases in the southern countries. T. cruzi I has shown great genetic diversity showing plausible subdivisions needed for this group. Recently, TcI has gained novel importance because of the lately discovered relation with cardiomyopathy manifestations that raises the importance of establishing subdivisions within this DTU Elsevier B.V. All rights reserved. Contents 1. Introduction T. cruzi I and first subdivisions Ecoepidemiology features and clinical trends of T. cruzi I Concluding remarks... 3 References Introduction Chagas disease caused by Trypanosoma cruzi is a complex systemic disease that affects over 10 million people in the American continent (WHO, 2007). The nomenclature of T. cruzi has recently been amended to reflect the high phenotypic and genotypic variability of this parasite. Six Discrete Typing Units (DTUs) have been proposed based on various features of T. cruzi (Zingales et al., 2009). Since 1999, when an international consensus on the nomenclature of T. cruzi was reached for the first time (Anon, 1999), two completely different genetic lineages have been discussed. Through isozyme studies, lineage Tcl has been revealed to be a homogeneous genetic group with dimorphisms in the mini-exon gene and in the divergent domain of the rdna of parasites (Miles et al., 1977; Souto et al., 1996; Tibayrenc and Ayala, 1988; Zingales et al., 1998). After ten years of research aimed at the verification of Tcl homogeneity, several molecular markers have demonstrated the existence of considerable genetic variability within T. cruzi I. Of note, the Corresponding author. Tel.: ; fax: address: fguhl@uniandes.edu.co (F. Guhl). first classification of T. cruzi I was a lineage linked to the sylvatic transmission cycle of Chagas disease. Since then, a large number of molecular markers have shown high genetic variability in T cruzi I (Cura et al., 2010; Falla et al., 2009; Herrera et al., 2007, 2009a,b; Llewellyn et al., 2009a, 2011; Mejía-Jaramillo et al., 2009a; Ocaña- Mayorga et al., 2010; Salazar et al., 2006; Spotorno et al., 2008; Triana et al., 2006). 2. T. cruzi I and first subdivisions Pioneering work by Brisse et al. (2001) presented trees constructed using Random Amplified Polymorphic DNA (RAPD) and Multi Locus Enzyme Electrophoresis (MLEE) analyses, and these demonstrated a significant amount of polytomies in the Tcl group. Barnabé et al., 2000 analysed a significant set of T. cruzi stocks from a wide ecogeographical range demonstrating the high genetic variability in T. cruzi based on 22 loci using MLEE, in this tree the branches within TcI shows a divergent topology suggesting the first attempts in the path to elucidate the genetic diversity within this formerly neglected DTU. Other reports from Colombia and Venezuela uncovered a large number of isolated groups of Tcl using techniques such as MLEE (Montilla et al., 2002; Saravia X/$ see front matter 2011 Elsevier B.V. All rights reserved. doi: /j.actatropica

3 2 F. Guhl, J.D. Ramírez / Acta Tropica 119 (2011) 1 4 et al., 1987; Widmer et al., 1984). Later, the analysis of data from LSSP-PCR and karyotyping of the variable region of kinetoplast DNA (kdna) demonstrated an association between genetic variation and the geographical origin of isolates (Salazar et al., 2006; Triana et al., 2006). These two studies revealed that Tcl is not homogeneous; however, because of the high number of copies of kdna and genetic divergence of each copy, this assessment was not robust enough. According to the results published by Fernandes et al. (1998, 2001) and O Oconnor et al. (2007), the intergenic region of the mini-exon gene was determined to be a suitable marker for the assessment of TcI genetic variability. In 2007, four genotypes formerly named haplotypes present in Colombian isolates of Tcl were reported, along with their relationship with the transmission cycles of Chagas disease (Herrera et al., 2007, 2009b). Genotype Ia, which is associated with human infection and domestic vectors, is present in a specific pattern at positions 28 with an Adenine and a motif TGTGTG at positions 35 40; genotype Ib, which is associated with human infection and peridomiciliary vectors, contains a T-C substitution at position 44; genotype Ic, which is not very robust due to the low number of isolates correlated with domiciliary vectors, is characterised by a TATATA sequence at position and genotype Id, which is associated with the sylvatic cycle is characterised by a deletion of nine nucleotides at positions of the microsatellite region of the mini-exon gene. According to the characteristics and the insertions, deletions and SNPs identified for each genotype, specific primers were developed to allow for the differential identification of three of the four genotypes (Ia, Ib and Id) (Falla et al., 2009). This work pioneered the study of genetic diversity of the Tcl group and prompted the scientific community to continue the search for other molecular markers to corroborate the genotypes of Tcl identified using the mini-exon gene. Subsequent work using the cytochrome b gene revealed the presence of two phylogenetically robust subgroups. The first subgroup is associated with human infection and vectors from Chile, Brazil and Colombia, whereas the second subgroup is mainly associated with caviomorph reservoirs, demonstrating the importance of these mammals in the evolution of trypanosomes (Spotorno et al., 2008). In 2009, Llewelyn et al. reported 135 TcI samples from various geographical regions endemic for Chagas disease in Latin America. The samples were subjected to microsatellite analysis and also demonstrated that the TcI group was highly diverse besides the work developed in Ecuador using a significant set of TcI isolates unravelling the genetic structure of TcI featuring two genotypes associated to the domestic/peridomestic cycle and the sylvatic cycle of transmission (Ocaña-Mayorga et al., 2010). In Brazil, a new TcI genotype has been described as TcBat due to its relatedness with bats from the genus Myotis and Noctilio based on Histone 2B, SSU rdna and Cytb (Marcili et al., 2009a). This genotype is very related to TcI and also to the hybrid DTUs TcIII and TcIV when the concatenated analysis was developed. This suggest and corroborates the high genetic diversity displayed by TcI taxon and could show clues about the evolutionary history of T. cruzi I with the host-relationships features. Fig. 1. Geographical distribution of Trypanosoma cruzi I genotypes in the Americas based on (Cura et al., 2010; Llewellyn et al., 2009a; Herrera et al., 2009b). Both sequence variation and microsatellite markers demonstrate that genotypes involved in human infections in Venezuela and Colombia are distinct from sympatric sylvatic variants. This is intriguing as it was recently demonstrated that the main vector in the region, Rhodnius prolixus, demonstrates panmixia between domestic and sylvatic environments across the western endemic region in Venezuela (Fitzpatrick et al., 2008). In 2009, when the nomenclature of T. cruzi was revised for the second time (Zingales et al., 2009), no divisions in TcI were created, based on the argument that studies with other markers should be completed. Recently, Cura et al. (2010) analysed a set of 105 Tcl samples from most of the endemic regions of Latin America and North America and confirmed the previously reported Ia, Ib, Ic and Id genotypes. Cura et al. also reported the existence of genotype Ie, which can be found in Bolivia, Argentina and Chile and is characterised by a 44-bp motif in the intergenic region of the mini-exon gene detected using specific primers designed for this purpose. In addition, it was determined that this variant is closely related to domestic cycles in Argentina, Bolivia and the sylvatic cycles of Chile, where Mepraia gajardoi and Mepraia spinolai play an important role in the transmission cycle of Chagas disease (Fig. 1). All these studies were pioneers in elucidating the genetic variability within TcI (Table 1). Table 1 Trypanosoma cruzi I subdivisions reported in the literature based on different molecular markers. Refs. Molecular marker Number of subdivisions Association of subdivision Geographical setting Salazar et al. (2006) LSSP-PCR (kdna) Geographical clustering Colombia Herrera et al. (2007) Mini-exon gene 4 Transmission cycles Colombia Spotorno et al. (2008) Cytochrome b gene 2 Transmission cycles Chile Herrera et al. (2009b) Mini-exon gene 5 Transmission cycles America Llewellyn et al. (2009a) Microsatellites 2 Transmission cycles America Marcili et al. (2009b) Histone 2B, SSU rdna and Cytb 2 Hosts (TcI and TcBat) Brazil Cura et al. (2010) SL-IR Mini-exon gene 5 Transmission cycles America

4 F. Guhl, J.D. Ramírez / Acta Tropica 119 (2011) Ecoepidemiology features and clinical trends of T. cruzi I The distribution of T. cruzi genotypes and reservoirs has important implications for the divisions of Tcl. In the southern part of the continent, infection of Canis familiaris has been associated with TcIV, V and VI, whereas infections in the north are associated with genotypes Ia and Ib (Falla et al., 2009; Herrera et al., 2009b). Furthermore, a significant number of Didelphis marsupialis in Colombia are infected with the TcId genotype, which suggests an association with the sylvatic transmission cycle. Similar studies in primates have demonstrated that TcI predominantly infects arboreal reservoirs (Cura et al., 2010; Falla et al., 2009). There are several hypotheses regarding the distribution of the different genetic groups of T. cruzi, suggesting that those reservoirs belonging to the arboreal ecotopes are preferentially infected with TcI and that terrestrial ecotopes are infected with TcII-TcVI (Yeo et al., 2005). This hypothesis is controversial in light of recent reports demonstrating that the arboreal ecotope reservoirs Monodelphis brevicaudata, Philander frenata and Didelphis aurita are infected with TcIII, TcIV and TcII, respectively (Marcili et al., 2009b; Llewellyn et al., 2009b). However, the associations are not absolute, and in the case of TcI, there was no apparent clustering of particular TcI genotypes with Didelphis in comparison to isolates from other arboreal mammals (Llewellyn et al., 2009b). Also for phylogeographical analyses of TcIII, the results indicate that isolates cluster according to geography rather than host association (Marcili et al., 2009b; Llewellyn et al., 2009b). This could also be supported for the recent analysis developed in mammals naturally infected with TcI using microsatellites markers revealing the role of the mammals reservoirs in the diversifying selection of T. cruzi (Llewellyn et al., 2011). Two interesting studies of host response to different strains have confirmed, by comparative artificial infection, that in the southern USA two species of opossum (Monodelphis domestica and Didelphis virginiana) seem to be resistant to TcIV (Roellig et al., 2009a; Roellig et al., 2009b). This highlights a mechanism for the association of a vertebrate host with one strain over others. The strong association between TcI and Rhodnius species can also be explained by a similar mechanism: comparative artificial infection studies of R. prolixus with various strains revealed a tendency for it to be resistant to infection by TcII (Mello et al., 1996). For triatomines, susceptibility or resistance to trypanosome infections seems to be modulated by the intestinal symbionts, which are vital for development (Azambuja et al., 2005). T. cruzi is considered to be subpathogenic to triatomines, whereas Trypanosoma rangeli is another species that commonly infects Rhodnius species and causes pathogenicity based on a reduction of the number of symbionts (Vallejo et al., 2009). Some studies using different species of triatomines such as Rhodnius pallescens, Triatoma dimidiata, Rhodnius colombiensis and Panstrongylus geniculatus have shown the affinity of TcI to infect these species in comparison with TcII (Mejía- Jaramillo et al., 2009b). A study of four Rhodnius species artificially infected with different strains of T. rangeli showed a measurable difference in response and indicated adaptation of trypanosome strains to the local vector species (Machado et al., 2001). At least half of all species of triatomine bugs have been found naturally infected with T. cruzi (Lent and Wygodzinsky, 1979; Schofield, 1994). Unfortunately the vast majority of these records do not include specific strain associations. Clearly this is an area of potential research. In the context of dispersal triggered by starvation, there is evidence that starvation decreases T. cruzi infection in triatomines (Kollien and Schaub, 1998) and in some species starvation may clear infection altogether (Phillips and Bertram, 1967; Vargas and Zeledon, 1985). This factor could go towards explaining paradigms such as in Venezuela where sylvatic and domestic bugs seem to be in panmixia but TcI shows discrete general clustering of sylvatic and domestic cycles (Fitzpatrick et al., 2008; Llewellyn et al., 2009b). Triatomine bugs directly determine the etiology of the strains of T. cruzi involved in human transmission cycles. This is clear because despite TcI and Didelphis being widespread, it is the northern distribution of Rhodnius that corresponds with its occurrence in human cycles. Overall, the aspects of epidemiological relevance are that associations between terrestrial ecology, Triatoma infestans, terrestrial mammals, and T. cruzi strains TcII/TcIV have lead to the prominence of TcII, TcV, and TcIV in human infections in the southern region of South America. In the northern regions human American Trypanosomiasis infections seem to stem from TcI associated with arboreal Rhodnius and arboreal mammals. Molecular epidemiology studies of T. cruzi have attempted to establish the effects of different DTUs in the clinical progression of Chagas disease. Several studies have shown the effect of genetic variability on the host immune response (dos Santos et al., 2009; Melquiades-Rodriguez et al., 2010; Ramírez et al., 2009). It was previously known that cardiopathies in southern cone countries were caused by TcII, TcV and TcVI, but it has recently been demonstrated that TcI can play an important role specifically in severe cardiopathies related to Chagas disease. Studies of cardiac biopsies from Argentinean patients revealed that patients with severe myocarditis were infected with TcI, whereas those with moderate or absent myocarditis were infected with TcII, TcV or TcVI (Burgos et al., 2010). At the same time that the TcI genotype was found in the severe myocarditis patients, it was demonstrated that in patients with chronic chagasic cardiopathy, the TcIa genotype was most commonly found in the bloodstream, whereas TcId was most commonly found in cardiac biopsies. These results are consistent with reports from patients in Colombia, where the least and most prevalent Tcl genotypes in adult patients with chronic chagasic cardiopathy were Tcld and TcIa, respectively (Ramírez et al., 2010). This suggests a possible type of histotropism by Tcl genotypes and the epidemiological importance of this DTU in the southern countries, where cardiopathies were previously thought to be caused primarily by TcII, TcV and TcVI. 4. Concluding remarks Recent studies have confirmed the presence of the Tcl genotypes in Argentina, Brazil, Bolivia, Chile, Colombia, Mexico, Panama, Paraguay, French Guiana, Venezuela and the USA (Cura et al., 2010; Tomazini et al., 2010). Furthermore, intergenic region of mini-exon gene sequence studies have largely confirmed these genotypes in Bolivia, Mexico, Brazil and Argentina (Herrera et al., 2009a), which suggest that the TcIe genotype reported by Cura et al. (2010) may correspond to the Bolivian group reported by Herrera in Based on the intergenic region of the gene mini-exon, there is a wide distribution of genotypes of Tcl in the Americas, a premise that has been confirmed by Llewellyn et al. (2009a) using microsatellite markers in which there are specific genotypes associated with human infection in Bolivia, Brazil, Venezuela and Ocaña-Mayorga et al. (2010) in Ecuador. The wide genetic diversity of Tcl in the Americas is shown in Fig. 1 where has been established the geographical distribution of TcI genotypes along the Americas. The implications of these studies in the clinical manifestation of Chagas disease are important and suggest the need to pursue additional phylogeographic studies using other molecular markers to develop and implement strategies aimed at the mitigation of Chagas disease across the continent. After all these published works the question remains and arises, is it necessary a subdivision within TcI?, and should the nomenclature be revised?, maybe that is a question that probably will be answered in nine years where the third revision of the T. cruzi nomenclature will be held when we celebrate 110 years of the discovery of Chagas disease and maybe new molecular markers and phylogeographical data will help to solve this enigmatic question.

5 4 F. Guhl, J.D. Ramírez / Acta Tropica 119 (2011) 1 4 References Anonymous, Recommendations from a satellite meeting. Mem. Inst. Oswaldo Cruz 94, Azambuja, P., Ratcliffe, N.A., Garcia, E.S., Towards an understanding of the interactions of Trypanosoma cruzi and Trypanosoma rangeli within the reduviid insect host Rhodnius prolixus. Ann. Acad. Brasil. Ciên. 77, Barnabé, C., Brisse, S., Tibayrenc, M., Population structure and genetic typing of Trypanosoma cruzi, the agent of Chagas disease: a multilocus enzyme electrophoresis approach. Parasitology 120, Burgos, J.M., Diez, M., Vigliano, C., et al., Molecular identification of Trypanosoma cruzi Discrete Typing Units in end-stage chronic Chagas heart disease and reactivation after heart transplantation. Clin. Infect. Dis., 51. Brisse, S., Verhoef, J., Tibayrenc, M., Characterisation of large and small subunit rrna and mini-exon genes further supports the distinction of six Trypanosoma cruzi lineages. Int. J. Parasitol. 36, dos Santos, D.M., Talvani, A., da Mata Guedes, P.M., et al., Trypanosoma cruzi: genetic diversity influences the profile of immunoglobulins during experimental infection. Exp. Parasitol. 121, Cura, C.I., Mejia-Jaramillo, A.M., Duffy, T., et al., Trypanosoma cruzi I genotypes indifferent geographc regions and transmission cycles based on a microsatellitemotif of the ispliced leader genes. Int. J. Parasitol. 40, Falla, A., Herrera, C., Fajardo, A., et al., Haplotype identification within Trypanosoma cruzi I in Colombian isolates from several reservoirs, vectors and humans. Acta Trop 110, Fernandes, O., Sturm, N.R., Derré, R., et al., The mini-exon gene: a genetic marker for zymodeme III of Trypanosoma cruzi. Mol. Biochem. Parasitol. 95, Fernandes, O., Santos, S.S., Copulillo, E., et al., A mini-exon multiplex polymerase chain reaction to distinguish the major groups of Trypanosoma cruzi and T. rangeli. Braz. Amazon. Trans. Roy. Soc. Trop. Med. Hyg. 95, Fitzpatrick, S., Feliciangeli, M.D., Sanchez-Martin, M.J., et al., Molecular genetics reveal that sylvatic Rhodnius prolixus do colonise rural houses. PLoS Negl. Trop. Dis. 2. Herrera, C., Bargues, M.D., Fajardo, A., et al., Identifying four Trypanosoma cruzi I isolate haplotypes from different geographic regions in Colombia. Infect. Genet. Evol. 7, Herrera, C.P., Guhl, F., Falla, A., et al., 2009a. Concordancia de haplotipos propuestos de Trypanosoma cruzi con aislamientos de otras regiones de Latinoamérica. Biomédica, 29. Herrera, C., Guhl, F., Falla, A., et al., 2009b. Genetic variability and phylogenetic relationships within Trypanosoma cruzi I isolated in Colombia based on miniexon gene sequences. J. Parasitol. Res., ID Kollien, A.H., Schaub, G.A., Development of Trypanosoma cruzi after starvation and feeding of the vector-a review. Tokai J. Exp. Clin. Med. 23, Lent, H., Wygodzinsky, P., Revision of the Triatominae, Hemiptera Reduviidae and their significance as vectors of Chagas disease. Bull. Am. Mus. Nat. Hist. 163, Llewellyn, M.S., Miles, M.A., Carrasco, H.J., et al., 2009a. Genome-Scale Multilocus Microsatellite Typing of Trypanosoma cruzi Discrete Typing Unit I revals phylogeographic structure and specific genotypes linked to human infection. PLoS Pathog., 5. Llewellyn, M.S., Lewis, M.D., Acosta, N., et al., 2009b. Trypanosoma cruzi IIc: phylogenetic and phylogeographic insights from sequence and microsatellite analysis and potential impact on emergent Chagas disease. PloS Negl. Trop. Dis. 3. Llewellyn, M., Rivett-Carnac, J.B., Fizpatrick, S., et al. (2011). Extraordinary Trypanosoma cruzi diversity within single mammalian reservoir hosts implies a mechanism of diversifying selection. Int. J. Parasitol., in press. Machado, E.M.M., Fernandes, A.J., Murta, S.M.F., et al., A study of experimental reinfection by Trypanosoma cruzi in dogs. Am. J. Trop. Med. Hyg. 65, Marcili, A., Lima, M., Cavazzana, M., et al., 2009a. A new genotype of Trypanosoma cruzi associated with bats evidenced by phylogenetic analyses using SSU rdna Cytochrome b and Histone H2B genes and genotyping based on ITS1 rdna. Parasitology 136, Marcili, A., Lima, L., Valente, V.C., et al., 2009b. Comparative phylogeography of Trypanosoma cruzi TCIIc: new hosts, association with terrestrial ecotopes and spatial clustering. Inf. Gen. Evol. 9, Mejía-Jaramillo, A.M., Arboleda-Sánchez, S., Rodríguez, I.B., et al., 2009a. Geographical clustering of Trypanosoma cruzi I groups from Colombia revealed by low-stringency single specific primer-pcr of the intergenic regions of splicedleader genes. Parasitol. Res. 104, Mejía-Jaramillo, A.M., Peña, V.H., Triana-Chávez, O., 2009b. Trypanosoma cruzi: biological characterization of lineages I and II supports the predominance of lineage I in Colombia. Exp. Parasitol. 121, Miles, M.A., Toye, P.J., Oswald, S.C., et al., The identification by isoenzyme patterns of two distinct strain-groups of Trypanosoma cruzi, circulating independently in a rural area of Brazil. Trans. R. Soc. Trop. Med. Hyg. 71, Montilla, M.M., Guhl, F., Jaramillo, C., et al., Isoenzyme clustering of Trypanosomatidae Colombian populations. Am. J. Trop. Med. Hyg. 66, Mello, C.B., Azambuja, P., Garcia, E.S., et al., Differential in vitro and in vivo behavior of three strains of Trypanosoma cruzi in the gut and hemolymph of Rhodnius prolixus. Exp. Parasitol. 82, Melquiades-Rodriguez, C., Silva-Valadares, H., Fortes-Francisco, A., et al., Coinfection with different Trypanosoma cruzi strains interferes with the host immune response to infection. PLoS Negl. Trop. Dis. 4. Ocaña-Mayorga, S., Llewellyn, M., Costales, J., et al., Sex, subdivision and domestic dispersal of Trypanosoma cruzi lineage I in southern Ecuador. PLoS Negl. Trop. Dis. 4. O Oconnor, O., Bosseno, M.F., Barnabé, C., et al., Genetic clustering of Trypanosoma cruzi I lineage evidenced by intergenic miniexon sequencing. Inf. Gen. Evol. 7, Phillips, N.R., Bertram, D.S., Laboratory studies of Trypanosoma cruzi infections in Rhodnius prolixus-larvae and adults in Triatoma infestans, T. protracta and T. maculata-adults. J. Med. Entomol. 4, Ramírez, J.D., Guhl, F., Umezawa, E.S., et al., Evaluation of adult chronic Chagas heart disease diagnosis by molecular and serological methods. J. Clin. Microbiol. 47, Ramírez, J.D., Guhl, F., Rendon, L.M., et al., Chagas cardiomyopathy manifestations and Trypanosoma cruzi genotypes circulating in chronic chagasic patients. PloS Negl. Trop. Dis. 4. Roellig, D.M., Ellis, A.E., Yabsley, M.J., 2009a. Genetically different isolates of Trypanosoma cruzi elicit different infection dynamics in raccoons, Procyon lotor and Virginia opossums, Didelphis virginiana. Int. J. Parasitol. 39, Roellig, D.M., McMillan, K., Ellis, A.E., et al., 2009b. Experimental infection of two South American reservoirs with four distinct strains of Trypanosoma cruzi. Parasitology, 1 8. Salazar, A., Schijman, A.G., Triana, O., High variability of Colombian Trypanosoma cruzi lineage I stocks as revealed by low-stringency single primer-pcr minicircle signatures. Acta Trop. 100, Saravia, N.G., Holguín, A.F., Cibulskis, R.E., et al., Divergent isoenzymes profiles of sylvatic and domiciliary Trypanosoma cruzi in the eastern plains, piedmont and highlands of Colombia. Am. J. Trop. Med. Hyg. 36, Schofield, C.J., Triatominae-Biology & Control. Eurocommunica Publications, Bognor Regis, West Sussex, UK. Souto, R.P., Fernandes, O., Macedo, A.M., et al., DNA markers define two major phylogenetic lineages of Trypanosoma cruzi. Mol. Biochem. Parasitol. 83, Spotorno, A.E., Córdova, L., Solari, A., Differentiation of Trypanosoma cruzi I subgroups through characterization of cytochrome b gene sequences. Infect. Genet. Evol. 8, Tibayrenc, M., Ayala, F., Isoenzyme variability in Trypanosoma cruzi the agent of Chagas disease: genetical, taxonomical and epidemiological significance. Evolution 42, Triana, O., Ortiz, S., Dujardin, J.C., et al., Trypanosoma cruzi; Variability of stocks from Colombia determined by molecular karyotype and minicircle southern blot analysis. Exp. Parasitol. 113, Tomazini, N., Lauthier, J.F., Monje-Rumi, M., et al., Interest and limitations of SplicedLeader Intergenic Region sequences for analyzing Trypanosoma cruzi Iphylogenetic diversity in the Argentinean Chaco. Inf. Genet. Evol. 11, Vallejo, G.A., Guhl, F., Schaub, G.A., Triatominae-Trypanosoma cruzi/t. rangeli: vector parasite interactions. Acta Trop. 110, Widmer, G., Marinkelle, C.J., Guhl, F., et al., Isoenzyme profiles of Trypanosoma cruzi stocks from Colombia and Ecuador. Ann. Trop. Med. Parasitol. 79, Vargas, L.G., Zeledon, R., Effect of fasting on Trypanosoma cruzi infection in Triatoma dimidiata, Hemiptera: Reduviidae. J. Med. Entomol. 22, 683. World Health Organization, WHO Special Programme for Research and Training in Tropical Diseases, TDR. Report of scientific group in Chagas disease. Buenos Aires, Argentina, April 17 20, Update July Yeo, M., Acosta, N., Llewellyn, M., et al., Origins of Chagas disease: Didelphis species are natural hosts of Trypanosoma cruzi I and armadillos hosts of Trypanosoma cruzi II, including hybrids. Int. J. Parasitol. 35, Zingales, B., Souto, R.P., Mangia, R.H., et al., Molecular epidemiology of American tripanosomiasis on Brazil based on dimorphisms of rrna and mini-exon gene sequences. Int. J. Parasitol. 28, Zingales, B., Andrade, S.G., Briones, M.R.S., et al., A new consensus for Trypanosoma cruzi intraespecific nomenclature: second revision meeting recommends TcI to TcVI. Mem. Inst. Oswaldo Cruz 104,