Identification and lineage genotyping of South American trypanosomes using fluorescent fragment length barcoding P.B. Hamilton a, *, M.D. Lewis b , C. Cruickshank a , M.W. Gaunt b , M. Yeo b , M.S. Llewellyn b , S.A. Valente c , F. Maia da Silva d , J.R. Stevens a , M.A. Miles b , M.M.G. Teixeira d a School of Q1 Biosciences, College of Life and Environmental Sciences, University of Exeter, Prince of Wales Road, Exeter EX4 4PS, United Kingdom b Pathogen Molecular Biology Unit, Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E 7HT, United Kingdom c Instituto Evandro Chagas, Bele ´m, PA 67030-070, Brazil d Departamento de Parasitologia, Instituto de Cieˆncias Biome ´dicas, Universidade de Sa˜o Paulo, Sa˜o Paulo, SP 05508-900, Brazil 1. Introduction Trypanosoma cruzi and Trypanosoma rangeli are the two species of human-infective trypanosomes occurring in overlapping areas of South and Central America. T. cruzi causes Chagas disease, a condition that affects at least 8 million people, with 100 million at risk and 14,000 deaths annually (Jannin and Salvatella, 2006). Despite recent advances in disrupting vector transmission in Southern Cone countries, this disease remains a major public health problem in Latin America (Schofield et al., 2006; Miles et al., 2009). In regions endemic for Chagas disease, T. cruzi circulates between humans and domestic animals and is transmitted by domiciliated triatomine bugs. However, infection by T. cruzi is a widespread zoonosis, ranging from the southern half of the USA to the southernmost countries of South America (Marcili et al., 2009c). T. rangeli is not believed to cause disease in humans. A high prevalence of T. rangeli in humans has been reported in Central America and northwestern South America, where concomitant infections and serological cross-reactivity with T. cruzi make diagnosis of Chagas disease difficult (Vallejo et al., 2009). Both T. cruzi and T. rangeli have a wide mammalian host range and are transmitted by a large diversity of triatomine bugs, although only species of the genus Rhodnius transmits T. rangeli (Maia da Silva et al., 2007; Vallejo et al., 2009). Molecular studies have revealed high genetic diversity in T. cruzi and T. rangeli, with isolates of both species distributed into several lineages, also called discrete taxonomic units (DTU) within T. cruzi (Stevens et al., 1999; Maia da Silva et al., 2007; Miles et al., 2009; Vallejo et al., 2009). At least six lineages of T. cruzi cruzi have been described using molecular markers including RAPDs, SSU Infection, Genetics and Evolution xxx (2010) xxx–xxx 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 ARTICLE INFO Article history: Received 20 August 2010 Received in revised form 14 October 2010 Accepted 15 October 2010 Available online xxx Keywords: Co-infection Genetic diversity Vector Chagas disease Protozoa Kinetoplastid ABSTRACT Trypanosoma cruzi and Trypanosoma rangeli are human-infective blood parasites, largely restricted to Central and South America. They also infect a wide range of wild and domestic mammals and are transmitted by a numerous species of triatomine bugs. There are significant overlaps in the host and geographical ranges of both species. The two species consist of a number of distinct phylogenetic lineages. A range of PCR-based techniques have been developed to differentiate between these species and to assign their isolates into lineages. However, the existence of at least six and five lineages within T. cruzi and T. rangeli, respectively, makes identification of the full range of isolates difficult and time consuming. Here we have applied fluorescent fragment length barcoding (FFLB) to the problem of identifying and genotyping T. cruzi, T. rangeli and other South American trypanosomes. This technique discriminates species on the basis of length polymorphism of regions of the rDNA locus. FFLB was able to differentiate many trypanosome species known from South American mammals: T. cruzi cruzi, T. cruzi marinkellei, T. dionisii-like, T. evansi, T. lewisi, T. rangeli, T. theileri and T. vivax. Furthermore, all five T. rangeli lineages and many T. cruzi lineages could be identified, except the hybrid lineages TcV and TcVI that could not be distinguished from lineages III and II respectively. This method also allowed identification of mixed infections of T. cruzi and T. rangeli lineages in naturally infected triatomine bugs. The ability of FFLB to genotype multiple lineages of T. cruzi and T. rangeli together with other trypanosome species, using the same primer sets is an advantage over other currently available techniques. Overall, these results demonstrate that FFLB is a useful method for species diagnosis, genotyping and understanding the epidemiology of American trypanosomes. ß 2010 Elsevier B.V. All rights reserved. * Corresponding author. Tel.: +44 01392 263917; fax: +44 01392 263700. E-mail address: p.b.hamilton@exeter.ac.uk (P.B. Hamilton). G Model MEEGID 858 1–8 Please cite this article in press as: Hamilton, P.B., et al., Identification and lineage genotyping of South American trypanosomes using fluorescent fragment length barcoding. Infect. Genet. Evol. (2010), doi:10.1016/j.meegid.2010.10.012 Contents lists available at ScienceDirect Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid 1567-1348/$ – see front matter ß 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.meegid.2010.10.012