Genome-Scale Multilocus Microsatellite Typing of Trypanosoma cruzi Discrete Typing Unit I Reveals Phylogeographic Structure and Specific Genotypes Linked to Human Infection Martin S. Llewellyn 1 *, Michael A. Miles 1 , Hernan J. Carrasco 2 , Michael D. Lewis 1 , Matthew Yeo 1 , Jorge Vargas 3 , Faustino Torrico 4 , Patricio Diosque 5 , Vera Valente 6 , Sebastiao A. Valente 6 , Michael W. Gaunt 1 1 London School of Hygiene and Tropical Medicine, London, United Kingdom, 2 Instituto de Medicina Tropical, Universidad Central de Venezuela, Los Chaguaramos, Caracas, Venezuela, 3 Centro Nacional de Enfermedades Tropicales, Santa Cruz, Bolivia, 4 Universidad Mayor de San Simon, Cochabamba, Bolivia, 5 Consejo Nacional de Investigaciones Cientı ´ficas y Te ´cnicas, Instituto de Patologia Experimental, Universidad Nacional de Salta, Salta, Argentina, 6 Instituto Evandro Chagas, Rodovia, Belem, Para, Brazil Abstract Trypanosoma cruzi is the most important parasitic infection in Latin America and is also genetically highly diverse, with at least six discrete typing units (DTUs) reported: Tc I, IIa, IIb, IIc, IId, and IIe. However, the current six-genotype classification is likely to be a poor reflection of the total genetic diversity present in this undeniably ancient parasite. To determine whether epidemiologically important information is ‘‘hidden’’ at the sub-DTU level, we developed a 48-marker panel of polymorphic microsatellite loci to investigate population structure among 135 samples from across the geographic distribution of TcI. This DTU is the major cause of resurgent human disease in northern South America but also occurs in silvatic triatomine vectors and mammalian reservoir hosts throughout the continent. Based on a total dataset of 12,329 alleles, we demonstrate that silvatic TcI populations are extraordinarily genetically diverse, show spatial structuring on a continental scale, and have undergone recent biogeographic expansion into the southern United States of America. Conversely, the majority of human strains sampled are restricted to two distinct groups characterised by a considerable reduction in genetic diversity with respect to isolates from silvatic sources. In Venezuela, most human isolates showed little identity with known local silvatic strains, despite frequent invasion of the domestic setting by infected adult vectors. Multilocus linkage indices indicate predominantly clonal parasite propagation among all populations. However, excess homozygosity among silvatic strains and raised heterozygosity among domestic populations suggest that some level of genetic recombination cannot be ruled out. The epidemiological significance of these findings is discussed. Citation: Llewellyn MS, Miles MA, Carrasco HJ, Lewis MD, Yeo M, et al. (2009) Genome-Scale Multilocus Microsatellite Typing of Trypanosoma cruzi Discrete Typing Unit I Reveals Phylogeographic Structure and Specific Genotypes Linked to Human Infection. PLoS Pathog 5(5): e1000410. doi:10.1371/ journal.ppat.1000410 Editor: Edward C. Holmes, The Pennsylvania State University, United States of America Received January 30, 2009; Accepted April 1, 2009; Published May 1, 2009 Copyright: ß 2009 Llewellyn et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding was provided by a Wellcome Trust junior research fellowship (MWG), The European Union Seventh Framework Programme rant 223034 (MAM), The Dr. Gordon-Smith Scholarship (MSL), The Swire Charitable Trust (MSL), The De Laszlo Foundation (MSL), and FONACIT (Venezuela) project G-2005000827 (HJC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: martin.llewellyn@lshtm.ac.uk Introduction T. cruzi, the etiological agent of Chagas disease, is a vector borne zoonosis and considered the most important parasitic infection in Latin America. In excess of 10 million people are thought to carry the parasite, with ten times that number at risk (http://www.who. int). Consistent with a long history on the continent [1], T. cruzi ecology in the silvatic environment is highly complex. Over 73 mammalian genera and just over half of 137 described species of haematophagous triatomine bug are involved with parasite carriage and transmission [2,3]. T. cruzi has an endemic range that stretches from the Southern USA to Northern Argentina. Most human infection is found in Central and South America and occurs primarily through contact with the contaminated faeces of domiciliated triatomine vector species. Genotypic data support the existence of six stable discrete typing units (DTUs) in T. cruzi: TcI, TcIIa, TcIIb, TcIIc, TcIId, and TcIIe [4]. Greatest molecular divergence is observed between TcI and TcIIb [1,4]. TcIIa and TcIIc have distinct genotypes but their affinities to other DTUs are inadequately understood [4,5]. TcIId and TcIIe are hybrids, and have haplotypes shared across TcIIb and TcIIc [1,6]. The ecological and epidemiological relevance of different T. cruzi DTUs have been the subject of considerable debate. Using a retrospective analysis of all available genotype records, we recently showed that diversification in the silvatic environment is likely to be driven by ecological niche as well as host species, with arboreal Didelphimorpha (opossums) the principal hosts of TcI, and terrestrial Cingulata (armadillos) the principal hosts of TcIIc [7]. TcI is a major agent for human disease north of the Amazon Basin [8,9], but is also ubiquitous in silvatic transmission cycles throughout the Americas [10,11]. In the Southern Cone region of South America, DTUs TcIIb, TcIId, and TcIIe cause most human infection [10]. With the exception of putative epizootic outbreaks [12], TcIIb, TcIId, and TcIIe are so far rare in the silvatic cycle [7]. PLoS Pathogens | www.plospathogens.org 1 May 2009 | Volume 5 | Issue 5 | e1000410