• View in gallery
    Figure 1.

    Example of LSU rDNA and mini-exon PCR product size polymorphism genotyping assay profiles. A, LSU rDNA—Lanes: 1, Rita cl5 (TcIIb); 2, Chaco23 col4 (TcIIb); 3, ARMA18 cl3 (TcIIc); 4, Sc43 cl1 (TcIId); 5, 92–80 cl2 (TcIId); 6, Bug2148 cl1 (TcIId), note absence of 125-bp band; 7, Chaco2 cl3 (TcIId); 8, PAH179 cl5 (TcIId); 9, Para6 cl4 (TcIId); 10, Para4 cl3 (TcIId); 11, Vinch101 cl1 (TcIId); 12, EPV20-1 cl1 (TcIIe); 13, P251 cl7 (TcIIe); 14, VFRA1 cl1 (TcIIe). B, LSU rDNA—Lanes: 1, C8 cl1 (TcI); 2, SAXP18 cl1 (TcI); 3, JR cl4 (TcI); 4, B187 cl10 (TcI); 5, 10R26 (TcIIa); 6, 92122102R [TcIIa(NA)]; 7, CanIII cl1 (TcIIa); 8, StC10R cl1 [TcIIa(NA)]; 9, Saimiri3 cl1 (TcIIa); 10, ERA cl2 (TcIIa); 11, JA2 cl2 (TcIIc); 12, SABP19 cl1 (TcIIc); 13, Vinch101 cl1 (TcIId); 14, LHVA cl4 (TcIIe). Note that comparison of Lanes 8–11 shows the four distinct product sizes: 130, 125, 117, and 110 bp. C, Mini-exon—Lanes: 1, M5631 cl5 (TcIIc); 2, JA2 cl2 (TcIIc); 3, ARMA18 cl3 (TcIIc); 4, 85/847 cl2 (TcIIc); 5, SABP cl1 (TcIIc); 6, VFRA1 cl1 (TcIIe); 7, Chaco2 cl3 (TcIId); 8, Esm cl3 (TcIIb); 9, X10/1 (TcI); 10, B187 cl10 (TcI); 11, JR cl4 (TcI); 12, 92101601P cl1 (TcI); 13, CanIII cl1 (TcIIa); 14, 92122102R [TcIIa(NA)]; 15, Saimiri3 cl1 (TcIIa); 16, X10610 cl5 (TcIIa); 17, ERA cl2 (TcIIa); 18, StC10R cl1 [TcIIa(NA)].

  • View in gallery
    Figure 2.

    Examples of PCR-RFLP genotyping profiles. A, HSP60: digestion products only are shown. Lanes: 1, X10610 cl5 (TcIIa); 2, Saimiri3 cl1 (TcIIa); 3, ERA cl2 (TcIIa); 4, JR cl4 (TcI); 5, 10R26 (TcIIa); 6, StC10R cl1 [TcIIa(NA)]; 7, CanIII cl1 (TcIIa); 8, X10/1 (TcI); 9, 92122102R [TcIIa(NA)]; 10, CJ005/PII (TcI); 11, B187 cl10 (TcI); 12, Chile C22 cl1 (TcI); 13, 92–80 cl2 (TcIId); 14, Rita cl5 (TcIIb); 15, Pot7a cl1 (TcIIb); 16, ARMA18 cl3 (TcIIc); 17, PAH179 cl5 (TcIId); 18, VFRA1 cl1 (TcIIe); 19, SABP19 cl1 (TcIIc); 20, M6241 cl6 (TcIIc); 21, Vinch101 cl1 (TcIId). B, Histone H1: digestion products from unpurified PCR products are shown. Lanes: 1, X10610 cl5 (TcIIa); 2, Saimiri3 cl1 (TcIIa); 3, ERA cl2 (TcIIa); 4, JR cl4 (TcIIc); 5, 10R26 (TcIIa); 6, StC10R cl1 [TcIIa(NA)]; 7, CanIII cl1 (TcIIa); 8, X10/1 (TcI); 9, Pot7b cl5 (TcIIb); 10, Rita cl5 (TcIIb); 11, JA2 cl2 (TcIIc); 12, ARMA13 cl1 (TcIIc); 13, CJ007/PI (TcI); 14, B187 cl10 (TcI); 15, SAXP18 cl1 (TcI); 16, 92122102R [TcIIa(NA)]; 17, SABP19 cl1 (TcIIc); 18, Para4 cl3 (TcIId); 19, CM25 cl2 (TcIIc); 20, PAH179 cl5 (TcIId); 21, Sc43 cl1 (TcIId); 22, Chaco17 col1 (TcIIe); 23, Tu18 cl2 (TcIIb); 24, Chaco23 col4 (TcIIb). C, GPI: each pair of lanes shows undigested PCR product followed by restriction digest products. Lanes: 1, SAXP18 cl1 (TcI); 2, VFRA1 cl1 (TcIIe); 3, StC10R cl1 [TcIIa(NA)]; 4, 10R26 (TcIIa); 5, Pot7a cl1 (TcIIb); 6, Rita cl5 (TcIIb); 7, JA2 cl2 (TcIIc); 8, Para4 cl3 (TcIId); 9, Vinch101 cl1 (TcIId); 10, Chaco9 col15 (TcIIe); 11, CanIII cl1 (TcIIa); 12, ARMA18 cl3 (TcIIc).

  • View in gallery
    Figure 3.

    Recommended triple-assay for discriminating T. cruzi DTUs.

  • 1

    WHO, 2002. World Health Report. Geneva: World Health Organisation.

  • 2

    Schofield CJ, Jannin J, Salvatella R, 2006. The future of Chagas disease control. Trends Parasitol 22 :583–588.

  • 3

    Dias JC, 2007. Southern Cone Initiative for the elimination of domestic populations of Triatoma infestans and the interruption of transfusion Chagas disease: historical aspects, present situation, and perspectives. Mem Inst Oswaldo Cruz 102 (Suppl 1):11–18.

    • Search Google Scholar
    • Export Citation
  • 4

    Feliciangeli MD, Sanchez-Martin MJ, Suarez B, Marrero R, Torrellas A, Bravo A, Medina M, Martinez C, Hernandez M, Duque N, Toyo J, Rangel R, 2007. Risk factors for Trypanosoma cruzi human infection in Barinas State, Venezuela. Am J Trop Med Hyg 76 :915–921.

    • Search Google Scholar
    • Export Citation
  • 5

    Gurtler RE, Kitron U, Cecere MC, Segura EL, Cohen JE, 2007. Sustainable vector control and management of Chagas disease in the Gran Chaco, Argentina. Proc Natl Acad Sci USA 104 :16194–16199.

    • Search Google Scholar
    • Export Citation
  • 6

    Coura JR, Junqueira ACV, Fernandes O, Valente SAS, Miles MA, 2002. Emerging Chagas disease in Amazonian Brazil. Trends Parasitol 18 :171–176.

    • Search Google Scholar
    • Export Citation
  • 7

    Dias JC, Bastos C, Araújo E, Mascarenhas AV, Martins Netto E, Grassi F, Silva M, Tatto E, Mendonça J, Araújo RF, Shikanai-Yasuda MA, Aras R, 2008. Acute Chagas disease outbreak associated with oral transmission. Rev Soc Bras Med Trop 41 :296–300.

    • Search Google Scholar
    • Export Citation
  • 8

    Nóbrega AA, Garcia MH, Tatto E, Obara MT, Costa E, Sobel J, Araujo WN, 2009. Oral transmission of Chagas disease by consumption of açaí palm fruit, Brazil. Emerg Infect Dis 5 :653–655.

    • Search Google Scholar
    • Export Citation
  • 9

    Fernandes O, Souto R, Castro J, Pereira J, Fernandes N, Junqueira A, Naiff R, Barrett T, Degrave W, Zingales B, Campbell D, Coura JR, 1998. Brazilian isolates of Trypanosoma cruzi from humans and triatomines classified into two lineages using mini-exon and ribosomal RNA sequences. Am J Trop Med Hyg 58 :807–811.

    • Search Google Scholar
    • Export Citation
  • 10

    Souto RP, Fernandes O, Macedo AM, Campbell DA, Zingales B, 1996. DNA markers define two major phylogenetic lineages of Trypanosoma cruzi. Mol Biochem Parasitol 83 :141–152.

    • Search Google Scholar
    • Export Citation
  • 11

    Oliveira RP, Broude NE, Macedo AM, Cantor CR, Smith CL, Pena SDJ, 1998. Probing the genetic population structure of Trypanosoma cruzi with polymorphic microsatellites. Proc Natl Acad Sci USA 95 :3776–3780.

    • Search Google Scholar
    • Export Citation
  • 12

    Valadares HMS, Pimenta JR, de Freitas JM, Duffy T, Bartholomeu DC, de Paula Oliveira R, Chiari E, Moreira MCV, Filho GB, Schijman AG, Franco GR, Machado CR, Pena SDJ, Macedo AM, 2008. Genetic profiling of Trypanosoma cruzi directly in infected tissues using nested PCR of polymorphic microsatellites. Int J Parasitol 38 :839–850.

    • Search Google Scholar
    • Export Citation
  • 13

    Llewellyn MS, Miles MA, Carrasco HJ, Lewis MD, Yeo M, Vargas J, Torrico F, Diosque P, Valente V, Valente SA, Gaunt MW, 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 :e1000410.

    • Search Google Scholar
    • Export Citation
  • 14

    Burgos JM, Altcheh J, Bisio M, Duffy T, Valadares HMS, Seidenstein ME, Piccinali R, Freitas JM, Levin MJ, Macchi L, Macedo AM, Freilij H, Schijman AG, 2007. Direct molecular profiling of minicircle signatures and lineages of Trypanosoma cruzi bloodstream populations causing congenital Chagas disease. Int J Parasitol 37 :1319–1327.

    • Search Google Scholar
    • Export Citation
  • 15

    Telleria J, Lafay B, Virreira M, Barnabe C, Tibayrenc M, Svoboda M, 2006. Trypanosoma cruzi: sequence analysis of the variable region of kinetoplast minicircles. Exp Parasitol 114 :279–288.

    • Search Google Scholar
    • Export Citation
  • 16

    Campbell D, Westenberger S, Sturm N, 2004. The determinants of Chagas disease: connecting parasite and host genetics. Curr Mol Med 4 :549–562.

    • Search Google Scholar
    • Export Citation
  • 17

    Momen H, 1999. Taxonomy of Trypanosoma cruzi: a commentary on characterization and nomenclature. Mem Inst Oswaldo Cruz 94 :181–184.

  • 18

    Tibayrenc M, 1998. Genetic epidemiology of parasitic protozoa and other infectious agents: the need for an integrated approach. Int J Parasitol 28 :85–104.

    • Search Google Scholar
    • Export Citation
  • 19

    Brisse S, Barnabe C, Tibayrenc M, 2000. Identification of six Trypanosoma cruzi phylogenetic lineages by random amplified polymorphic DNA and multilocus enzyme electrophoresis. Int J Parasitol 30 :35–44.

    • Search Google Scholar
    • Export Citation
  • 20

    Brisse S, Verhoef J, Tibayrenc M, 2001. Characterisation of large and small subunit rRNA and mini-exon genes further supports the distinction of six Trypanosoma cruzi lineages. Int J Parasitol 31 :1218–1226.

    • Search Google Scholar
    • Export Citation
  • 21

    Kawashita SY, Sanson GFO, Fernandes O, Zingales B, Briones MRS, 2001. Maximum-Likelihood divergence date estimates based on rRNA gene sequences suggest two scenarios of Trypanosoma cruzi intraspecific evolution. Mol Biol Evol 18 :2250–2259.

    • Search Google Scholar
    • Export Citation
  • 22

    Mendonça M, Nehme N, Santos S, Cupolillo E, Vargas N, Junqueira A, Naiff R, Barrett T, Coura J, Zingales B, Fernandes O, 2002. Two main clusters within Trypanosoma cruzi zymodeme 3 are defined by distinct regions of the ribosomal RNA cistron. Parasitology 124 :177–184.

    • Search Google Scholar
    • Export Citation
  • 23

    Ceballos LA, Cardinal MV, Vazquez-Prokopec GM, Lauricella MA, Orozco MM, Cortinas R, Schijman AG, Levin MJ, Kitron U, Gürtler RE, 2006. Long-term reduction of Trypanosoma cruzi infection in sylvatic mammals following deforestation and sustained vector surveillance in northwestern Argentina. Acta Trop 98 :286–296.

    • Search Google Scholar
    • Export Citation
  • 24

    Yeo M, Acosta N, Llewellyn M, Sanchez H, Adamson S, Miles GAJ, Lopez E, Gonzalez N, Patterson JS, Gaunt MW, de Arias AR, Miles MA, 2005. 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 :225–233.

    • Search Google Scholar
    • Export Citation
  • 25

    Fernandes O, Sturm NR, Derre R, Campbell DA, 1998. The mini-exon gene: a genetic marker for zymodeme III of Trypanosoma cruzi. Mol Biochem Parasitol 95 :129–133.

    • Search Google Scholar
    • Export Citation
  • 26

    Rozas M, Doncker SD, Adaui V, Coronado X, Barnabe C, Tibyarenc M, Solari A, Dujardin J-C, 2007. Multilocus polymerase chain reaction restriction fragment-length polymorphism genotyping of Trypanosoma cruzi (Chagas Disease): taxonomic and clinical applications. J Infect Dis 195 :1381–1388.

    • Search Google Scholar
    • Export Citation
  • 27

    Westenberger SJ, Barnabe C, Campbell DA, Sturm NR, 2005. Two hybridization events define the population structure of Trypanosoma cruzi. Genetics 171 :527–543.

    • Search Google Scholar
    • Export Citation
  • 28

    Lewis MD, Llewellyn MS, Gaunt MW, Yeo M, Carrasco HJ, Miles MA, 2009. Flow cytometric analysis and microsatellite genotyping reveal extensive DNA content variation in Trypanosoma cruzi populations and expose contrasts between natural and experimental hybrids. Int J Parasitol 39 :1305–1317.

    • Search Google Scholar
    • Export Citation
  • 29

    Carrasco H, Frame I, Valente S, Miles M, 1996. Genetic exchange as a possible source of genomic diversity in sylvatic populations of Trypanosoma cruzi. Am J Trop Med Hyg 54 :418–424.

    • Search Google Scholar
    • Export Citation
  • 30

    Sturm NR, Vargas NS, Westenberger SJ, Zingales B, Campbell DA, 2003. Evidence for multiple hybrid groups in Trypanosoma cruzi. Int J Parasitol 33 :269–279.

    • Search Google Scholar
    • Export Citation
  • 31

    Gaunt MW, Yeo M, Frame IA, Stothard JR, Carrasco HJ, Taylor MC, Mena SS, Veazey P, Miles GAJ, Acosta N, de Arias AR, Miles MA, 2003. Mechanism of genetic exchange in American trypanosomes. Nature 421 :936–939.

    • Search Google Scholar
    • Export Citation
  • 32

    Brisse S, Henriksson J, Barnabe C, Douzery EJP, Berkvens D, Serrano M, De Carvalho MRC, Buck GA, Dujardin J-C, Tibayrenc M, 2003. Evidence for genetic exchange and hybridization in Trypanosoma cruzi based on nucleotide sequences and molecular karyotype. Infect Genet Evol 2 :173–183.

    • Search Google Scholar
    • Export Citation
  • 33

    Machado CA, Ayala FJ, 2001. Nucleotide sequences provide evidence of genetic exchange among distantly related lineages of Trypanosoma cruzi. Proc Natl Acad Sci USA 98 :7396–7401.

    • Search Google Scholar
    • Export Citation
  • 34

    de Freitas JM, Augusto-Pinto L, Pimenta JR, Bastos-Rodrigues L, Gonçalves VF, Teixeira SMR, Chiari E, Junqueira AnCV, Fernandes O, Macedo AM, Machado CR, Pena SDJ, 2006. Ancestral genomes, sex, and the population structure of Trypanosoma cruzi. PLoS Pathog 2 :24.

    • Search Google Scholar
    • Export Citation
  • 35

    Barnabé C, Yaeger R, Pung O, Tibayrenc M, 2001. Trypanosoma cruzi: a considerable phylogenetic divergence indicates that the agent of Chagas disease is indigenous to the native fauna of the United States. Exp Parasitol 99 :73–79.

    • Search Google Scholar
    • Export Citation
  • 36

    Marcili A, Lima L, Valente VC, Valente SA, Batista JS, Junqueira AC, Souza AI, da Rosa JA, Campaner M, Lewis MD, Llewellyn MS, Miles MA, Teixeira MM, 2009. Comparative phylogeography of Trypanosoma cruzi TCIIc: new hosts, association with terrestrial ecotopes, and spatial clustering. Infect Genet Evol (In press).

    • Search Google Scholar
    • Export Citation
  • 37

    Thomas S, Westenberger SJ, Campbell DA, Sturm NR, 2005. Intragenomic spliced leader RNA array analysis of kineto-plastids reveals unexpected transcribed region diversity in Trypanosoma cruzi. Gene 352 :100–108.

    • Search Google Scholar
    • Export Citation
  • 38

    Herrera C, Bargues MD, Fajardo A, Montilla M, Triana O, Vallejo GA, Guhl F, 2007. Identifying four Trypanosoma cruzi I isolate haplotypes from different geographic regions in Colombia. Infect Genet Evol 7 :535–539.

    • Search Google Scholar
    • Export Citation
  • 39

    Mauricio IL, Yeo M, Baghaei M, Doto D, Pratlong F, Zemanova E, Dedet J-P, Lukes J, Miles MA, 2006. Towards multilocus sequence typing of the Leishmania donovani complex: resolving genotypes and haplotypes for five polymorphic metabolic enzymes (ASAT, GPI, NH1, NH2, PGD). Int J Parasitol 36 :757–769.

    • Search Google Scholar
    • Export Citation
  • 40

    Ravel C, Cortes S, Pratlong F, Morio F, Dedet J-P, Campino L, 2006. First report of genetic hybrids between two very divergent Leishmania species: Leishmania infantum and Leishmania major. Int J Parasitol 36 :1383–1388.

    • Search Google Scholar
    • Export Citation
  • 41

    Cooper MA, Adam RD, Worobey M, Sterling CR, 2007. Population genetics provides evidence for recombination in Giardia. Curr Biol 17 :1984–1988.

    • Search Google Scholar
    • Export Citation
  • 42

    Boyle JP, Rajasekar B, Saeij JPJ, Ajioka JW, Berriman M, Paulsen I, Roos DS, Sibley LD, White MW, Boothroyd JC, 2006. Just one cross appears capable of dramatically altering the population biology of a eukaryotic pathogen like Toxoplasma gondii. Proc Natl Acad Sci USA 103 :10514–10519.

    • Search Google Scholar
    • Export Citation
  • 43

    Dodgson AR, Pujol C, Pfaller MA, Denning DW, Soll DR, 2005. Evidence for recombination in Candida glabrata. Fungal Genet Biol 42 :233–243.

    • Search Google Scholar
    • Export Citation
  • 44

    Tavanti A, Gow NAR, Maiden MCJ, Odds FC, Shaw DJ, 2004. Genetic evidence for recombination in Candida albicans based on haplotype analysis. Fungal Genet Biol 41 :553–562.

    • Search Google Scholar
    • Export Citation
  • 45

    Bosseno M-F, Telleria J, Vargas F, Yaksic N, Noireau F, Morin A, Brenière SF, 1996. Trypanosoma cruzi: study of the distribution of two widespread clonal genotypes in Bolivian Triatoma infestans vectors shows a high frequency of mixed infections. Exp Parasitol 83 :275–282.

    • Search Google Scholar
    • Export Citation
  • 46

    Cardinal MV, Lauricella MA, Ceballos LA, Lanati L, Marcet PL, Levin MJ, Kitron U, Gürtler RE, Schijman AG, 2008. Molecular epidemiology of domestic and sylvatic Trypanosoma cruzi infection in rural northwestern Argentina. Int J Parasitol 38 :1533–1543.

    • Search Google Scholar
    • Export Citation
  • 47

    Yeo M, Lewis MD, Carrasco HJ, Acosta N, Llewellyn M, da Silva Valente SA, de Costa Valente V, de Arias AR, Miles MA, 2007. Resolution of multiclonal infections of Trypanosoma cruzi from naturally infected triatomine bugs and from experimentally infected mice by direct plating on a sensitive solid medium. Int J Parasitol 37 :111–120.

    • Search Google Scholar
    • Export Citation
  • 48

    Souto RP, Vargas N, Zingales B, 1999. Trypanosoma rangeli: discrimination from Trypanosoma cruzi based on a variable domain from the large subunit ribosomal RNA gene. Exp Parasitol 91 :306–314.

    • Search Google Scholar
    • Export Citation
  • 49

    Miles MA, Yeo M, Gaunt MW, 2003. Genetic diversity of Trypanosoma cruzi and the epidemiology of Chagas disease. Kelly JM, ed. Molecular Mechanisms of Pathogenesis in Chagas Disease. New York: Kluwer Academic, 1–15.

  • 50

    Tibayrenc M, Ayala F, 1988. Isozyme variability of Trypanosoma cruzi, the agent of Chagas’ disease: genetical, taxonomical and epidemiological significance. Evolution 42 :277–292.

    • Search Google Scholar
    • Export Citation
  • 51

    Anonymous, 1999. Recommendations from a satellite meeting. Mem Inst Oswaldo Cruz 94 :429–432.

  • 52

    Roellig DM, Brown EL, Barnabe C, Tibayrenc M, Steurer FJ, Yabsley MJ, 2008. Molecular typing of Trypanosoma cruzi isolates, United States. Emerg Infect Dis 14 :1123–1125.

    • Search Google Scholar
    • Export Citation
Past two years Past Year Past 30 Days
Abstract Views 0 0 0
Full Text Views 522 350 48
PDF Downloads 179 98 12
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 

 

 

Genotyping of Trypanosoma cruzi: Systematic Selection of Assays Allowing Rapid and Accurate Discrimination of All Known Lineages

Michael D. LewisPathogen Molecular Biology Unit, Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom; Instituto de Medicina Tropical, Facultad de Medicina, Universidad Central de Venezuela, Caracas, Venezuela

Search for other papers by Michael D. Lewis in
Current site
Google Scholar
PubMed
Close
,
Jonathan MaPathogen Molecular Biology Unit, Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom; Instituto de Medicina Tropical, Facultad de Medicina, Universidad Central de Venezuela, Caracas, Venezuela

Search for other papers by Jonathan Ma in
Current site
Google Scholar
PubMed
Close
,
Matthew YeoPathogen Molecular Biology Unit, Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom; Instituto de Medicina Tropical, Facultad de Medicina, Universidad Central de Venezuela, Caracas, Venezuela

Search for other papers by Matthew Yeo in
Current site
Google Scholar
PubMed
Close
,
Hernán J. CarrascoPathogen Molecular Biology Unit, Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom; Instituto de Medicina Tropical, Facultad de Medicina, Universidad Central de Venezuela, Caracas, Venezuela

Search for other papers by Hernán J. Carrasco in
Current site
Google Scholar
PubMed
Close
,
Martin S. LlewellynPathogen Molecular Biology Unit, Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom; Instituto de Medicina Tropical, Facultad de Medicina, Universidad Central de Venezuela, Caracas, Venezuela

Search for other papers by Martin S. Llewellyn in
Current site
Google Scholar
PubMed
Close
, and
Michael A. MilesPathogen Molecular Biology Unit, Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom; Instituto de Medicina Tropical, Facultad de Medicina, Universidad Central de Venezuela, Caracas, Venezuela

Search for other papers by Michael A. Miles in
Current site
Google Scholar
PubMed
Close

Trypanosoma cruzi, the agent of Chagas disease, can be subdivided into six discrete typing units (DTUs), TcI, TcIIa, TcIIb, TcIIc, TcIId or TcIIe, each having distinct epidemiologically important features. Dozens of genetic markers are available to determine the DTU to which a T. cruzi isolate belongs, but there is no consensus on which should be used. We selected five assays: three polymerase chain reaction (PCR)-restriction fragment length polymorphisms based on single nucleotide polymorphisms (SNPs) in the HSP60, Histone H1, and GPI loci, and PCR product size polymorphism of the LSU rDNA and mini-exon loci. Each assay was tested for its capacity to differentiate between DTUs using a panel of 48 genetically diverse T. cruzi clones. Some markers allowed unequivocal identification of individual DTUs, however, only by using a combination of multiple markers could all six DTUs be resolved. Based upon the results we recommend a triple-assay comprising the LSU rDNA, HSP60 and GPI markers for reliable, rapid, low-cost DTU assignment.

INTRODUCTION

The protozoan parasite Trypanosoma cruzi, causative agent of Chagas disease, is harbored by at least 10 million people in Latin America and is estimated to cause ~13,000 deaths per year.1 T. cruzi is endemic across the vast majority of Latin America and into the southern states of the United States, but Chagas disease occurs primarily in areas where human populations come into contact with domiciliated triatomine vector species. Furthermore, blood transfusion and congenital transmission can lead to cases of Chagas disease including cases outside Latin America. Control campaigns have resulted in reduced levels of T. cruzi transmission across much of the endemic area, yet significant challenges remain. 2,3 These include re-infestation of houses by vector species 4,5 and outbreaks associated with oral transmission caused by triatomine contamination of foods and drinks.68

Trypanosoma cruzi shows extremely high levels of genetic diversity, and a plethora of genetic markers can be used to stratify the species into various subdivisions, with greater or lesser levels of resolution depending on the markers used. Typing of genetic polymorphisms at relatively conserved loci can define major genetic subdivisions, 9,10 whereas analysis of highly variable loci such as microsatellites 1113 or kDNA minicircle sequences 14,15 allows higher level resolution, potentially even to the level of profiles that are specific to individual strains.

An understanding of the genetic diversity of any microbial pathogen is crucial, especially for epidemiologic research and for diagnostic, evolutionary, and basic biological studies. Historically, study of T. cruzi genetic diversity has been hampered by a lack of standardized typing methods and the use of various alternative nomenclatures 16,17 (Table 1). In some cases, this has led to confusion in the literature and made comparison between different studies problematic. For the purposes of molecular epidemiology, a useful conceptual development has been that of the discrete typing unit (DTU), which groups strains on the basis of shared characteristics of multilocus genotypes but without making explicit assumptions about their evolutionary relatedness. 18 For T. cruzi, multilocus genotyping has consistently shown six distinct DTUs, TcI, TcIIa, TcIIb, TcIIc, TcIId, and TcIIe, 19,20 each having distinct epidemiologic and evolutionary aspects. Although many typing systems are in use for T. cruzi, there is a lack of data regarding comparison of different methods, particularly with respect to the relatively undersampled DTUs TcIIa and TcIIc, which are only occasionally present in domestic settings.

Two of the most commonly used T. cruzi genotyping assays exploit sequence variability in the D7 divergent domain of the 24Sα rRNA locus (LSU rDNA) and in the non-transcribed intergenic region of the SL-RNA (mini-exon) array. This permits discrimination of some of the different lineages by simple visualization of differences in polymerase chain reaction (PCR) product size 9,10,2022 (Table 2). Using the rDNA target, some lineages are easily genotyped because they give single band profiles, for example, 110 bp for TcI/IIc or 125 bp for TcIIb/IIe. TcIId strains are typically characterized by the presence of both of these bands, although the larger band can be weak or absent entirely. 10,20 Furthermore, TcIIa does not have a single characteristic band size; some isolates, including the reference strain CanIII cl1, give a band smaller than 125 bp, estimated to be either 120 20 or 117 bp, 21 whereas TcIIa isolates from North America [TcIIa(NA)] seem to be characterized by a 130 bp band. 20 Regarding the mini-exon, early studies showed a multiplex PCR assay easily differentiated TcI (350 bp) from TcIIb/IId/IIe (300 bp). 9,10 Using this assay to characterize ZIII isolates (TcIIa and TcIIc) has proven to be less straightforward; some authors report a lack of amplification, 20 whereas others have successfully amplified products of 400 bp for TcIIa and 250 bp for TcIIc. 23,24 Others recommend the use of modified protocols using lineage-specific primers to allow discrimination of ZIII isolates. 14,25

A number of PCR-restriction fragment length polymorphism (RFLP) protocols have been described, 26,27 but to date, they have only been tested on a limited number of isolates, and it is not clear which are most suitable for standardized, widespread application. We selected three of these assays that, when used in combination, showed the potential to identify TcI, TcIIa, TcIIb, TcIIc, and a joint TcIId/TcIIe group. We set out to compare the performance of these three PCR-RFLP assays with the LSU rDNA and mini-exon genotyping assays using a large cohort of T. cruzi clones representing all six DTUs. We show that combining the LSU rDNA assay with two of the PCR-RFLP assays allows the simple, rapid, and low-cost resolution of all known T. cruzi DTUs.

MATERIALS AND METHODS

Parasite stocks and extraction of genomic DNA.

A panel of 48 T. cruzi biological clones representing all six DTUs was assembled (Table 3). They originate from diverse localities in endemic areas and consist of isolates from sylvatic and domestic transmission cycles; their sources include triatomine vectors, mammal hosts, and infected humans; full details of their origins are given elsewhere. 28 Parasites were cultivated in supplemented RPMI-1640 liquid medium (Sigma, Gillingham, UK) at 28°C as described previously. 29 Total genomic DNA was prepared from logarithmic phase cultures using standard phenol:chloroform protocols or alternatively using the Gentra Puregene Tissue Kit (Qiagen, Crawley, UK) according to the manufacturer’s protocols.

PCR product size polymorphism assays.

All strains were characterized by PCR amplification of the D7 divergent domain of the 24Sα rRNA gene (LSU rDNA) and the non-transcribed spacer of the mini-exon gene using standard protocols. 10,20 Amplification reactions contained 0.2 mmol/L of each dNTP, 1.5 mmol/L MgCl2, 1 pmol/μL of each primer, 1 Unit of Taq DNA polymerase (Bioline, London, UK) and 10–100ngg DNA. For the LSU rDNA, PCR primers D71 and D72 were used, and amplifications were performed using an initial denaturation step of 94°C for 3 minutes and then 27 amplification cycles (94°C for 1 minute, 60°C for 1 minute, 72°C for 1 minute), followed by a final elongation step at 72°C for 5 minutes. 10 For the mini-exon PCRs, a pool of three primers, TC, TC1, and TC2, was used, and amplifications were performed using an initial denaturation step of 94°C for 3 minutes and then 27 amplification cycles (94°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds), followed by a final elongation step at 72°C for 5 minutes. 10 LSU rDNA PCR products were separated by gel electrophoresis in 3% agarose gels and mini-exon products in 1.5% gels. For expected product sizes, see Table 2.

PCR-RFLP assays.

All strains were characterized by restriction enzyme digestion of PCR products from the amplification of three target loci, as first described by Westenberger and others, 27 with minor modifications to the protocol. The following target/restriction enzyme combinations were used: heat shock protein 60 (HSP60)/EcoRV, histone H1/AatII, and glucose-6-phosphate isomerase (GPI)/HhaI. Amplification reactions were as above but contained 2 mmol/L MgCl2, and the primer pairs used were as follows: HSP60_for and HSP60_rev (for HSP60),30 H1_for and H1_rev (for histone H1),30 and GPI_for and GPI_rev (for GPI).31 Amplifications were performed using a touchdown PCR strategy comprising an initial denaturation step of 3 minutes at 94°C, followed by four cycles (94°C for 30 seconds, 64°C for 30 seconds, 72°C for 1 minute), followed by 28 cycles (94°C for 30 seconds, 60°C for 30 seconds, 72°C for 1 minute), and then a final elongation step at 72°C for 10 minutes. PCR products were checked on 1.5% agarose gels and if necessary purified using the Qiaquick gel extraction kit (Qiagen) to remove non-specific products. For restriction enzyme digestion, 10 μL of PCR product (typically ~1 μg) was digested in a reaction containing 0.25 U/μL of the appropriate restriction enzyme, i.e., EcoRV (Promega, Southampton, UK), HhaI (NEB, Hitchin, UK), or AatII (NEB, Hitchin, UK), 100 ng/μL BSA, and 1× quantity of the manufacturer’s recommended reaction buffer in a total volume of 20 μL. The digestion reactions were incubated for 4 hours at 37°C, after which 5 μL of the reaction was used for restriction fragment size analysis using either 1.5% agarose gels (GPI/HhaI) or 3% gels (HSP60/EcoRV and histone H1/AatII). For expected product sizes, see Table 2.

RESULTS

LSU rDNA PCR product size polymorphism.

All samples were readily genotyped at this locus (Figure 1; Table 3). As expected, all TcI and TcIIc strains gave 110-bp bands and all TcIIb and TcIIe strains gave 125-bp bands. TcIId strains always had the 110-bp band; usually, the 125-bp band was also present, producing the characteristic double band profile. However, the intensity of the 125-bp band was variable across independent replicates and was sometimes not visible (Figure 1A). CanIII cl1 and three other TcIIa strains gave the expected intermediate-sized product (Figure 1B; Table 3), considered in this study to be 117 bp in accordance with Kawashita and others, 21 rather than 120 bp. 20 One South American TcIIa strain, Saimiri3 cl1, generated the 125-bp normally characteristic for TcIIb/IIe as shown previously. 20 The TcIIa(NA) strains gave 130-bp bands, as found previously for other strains of the same origin. 20 In summary, genotyping of this locus confirmed its general utility as a discriminatory marker for T. cruzi lineages; however, it does require very small differences in band size (≥ 5 bp) to be resolved, which can be technically challenging.

Mini-exon PCR product size polymorphism.

All samples were genotyped using PCR amplification of the mini-exon gene (Figure 1C; Table 3). In total, eight isolates were typed as TcIIc (250-bp products), 24 as TcIIb/d/e (300 bp), eleven as TcI (350 bp), and three as TcIIa (400 bp). Compared with the original multiple locus enzyme electrophoresis (MLEE) and/or random amplified polymorphic DNA (RAPD) genotyping, most strains gave product sizes as expected. One strain, Saimiri3 cl1, gave a product estimated to be 380 bp (Figure 1C); a previous study had recorded no amplification for this strain. 20 Another, 10R26, gave no amplification despite repeated attempts, although this strain has previously been reported to contain a TcI-like sequence. 30

It should be noted that for a number of the strains previously typed as TcIIc (Table 3), the mini-exon exhibited a lack of reproducibility across repeated experiments: the diagnostic 250-bp band was often faint, and multiple non-specific bands were observed more frequently compared with other strains. As an example, profiles showing bands at both 250 and 300 bp were observed for ARMA13 cl1 and SABP19 cl1 (Figure 1C; Table 3). Difficulty in genotyping TcIIc strains by mini-exon PCR is in keeping with previous findings. 20 Nevertheless, in this study, the diagnostic 250-bp band was not observed for any other strains and thus was considered to be indicative of a TcIIc genotype.

The TcIIa strains showed heterogeneity in the size of the mini-exon amplification product and also frequently presented non-specific bands. Besides the two strains already mentioned (10R26 and Saimiri3 cl1), CanIII cl1, 92122102R, and StC10R cl1 did give products of 400 bp, but X10610 cl5 and ERA cl2 produced bands of 350 bp, normally characteristic of TcI.

PCR-RFLPassays.

A recent study detailed the development of six PCR-RFLP assays for genotyping T. cruzi lineages.27 According to the data presented for a panel of 26 T. cruzi isolates (ten of which are also in our panel), there was no single assay that could split the strains into more than three groups of genotypes. However, by combining the data from three of these assays (HSP60 digested with EcoRV, histone H1 digested with Aat II, and GPI digested with HhaI), all DTUs except TcIId/IIe would be predicted to have unique multiple assay profiles. To validate the potential of this typing scheme, the three PCR-RFLP assays were applied to all samples.

HSP60. Minimal non-specific PCR products for HSP60 were observed, and this meant that the digestion reaction could be performed directly on PCR products without the need for purification, thus reducing the time needed for this assay. An example of RFLP profiles generated by this assay is shown in Figure 2. The genotype group designations (TcI/IIa/IIb, TcIIc, or TcIId/IIe) 27 were applied to the entire cohort of samples. Across the whole panel of T. cruzi, the genotype assignments showed an exact correlation with those predicted by other genotyping methods (Table 3). Notably, this assay proved to be a reliable method to discriminate TcIIc strains from all other lineages.

Histone H1. The PCR-RFLP of histone H1 was performed for all samples and produced the same three profiles (TcI/IIc, TcIIb, or TcIIa/IId/IIe) identified by Westenberger and others 27 (Figure 2; Table 2). Digestion of unpurified PCR products, in some cases, generated some non-specific bands, predominantly one ~320 bp in size. Such bands, however, did not hamper detection of the diagnostic bands. Comparison of the genotype assignments with expectations showed that 45 of 48 samples in this study gave RFLP profiles consistent with these expectations. Exceptions were Saimiri3 cl1, StC10R cl1, and 92122102R, all of which gave TcI/IIc profiles rather than the expected TcIIa/IId/IIe profile. This single assay reliably resolved DTU TcIIb from all others.

GPI. As for HSP60, the digestion reaction for GPI amplification products could be performed directly without the need for purification. All the T. cruzi isolates in this study gave RFLP profiles (Figure 2) that could easily be assigned to the possible genotype groups (TcI/IIc, TcIIa/IIb, or TcIId/IIe) identified by Westenberger and others. 27 Genotypes were consistent with those expected for each DTU based on other markers for 46 of 48 samples (Table 3). Exceptions were StC10R cl1 and 92122102R, both of which gave TcI/IIc profiles rather than the expected TcIIa/IIb profile.

DISCUSSION

For many years, MLEE was the method of choice for resolving T. cruzi subgroups. With the advent of direct genetic typing, a range of PCR-based assays capable of delineating T. cruzi subdivisions to varying extents were developed and readily applied. However, there has been a tendency for the number of loci used for typing to be reduced to only two that are widely used (LSU rDNA and mini-exon) or various additional loci that are only used by a small number of laboratories. This creates the problem of reduced discriminatory power and/or difficulty in comparing work in different laboratories using different typing systems. Although high-resolution genetic typing can now be achieved using multilocus sequence typing (MLST) 27,32,33 or multilocus microsatellite typing (MLMT), 11,13,34 these methods are impractical for simple DTU assignment. For that objective, PCR-RFLP assays hold much promise given the low resource requirement. We selected three PCR-RFLP assays based on data from an analysis of 26 strains showing that they had good potential for differentiating between T. cruzi DTUs.27 We set out to test the performance of these assays for typing a large cohort of cloned T. cruzi isolates and compared them to the commonly used mini-exon and LSU rDNA typing assays.

Typing of the LSU rDNA allowed discrimination of the following groups: TcI/TcIIc, TcIIb/TcIIe, and most TcIId samples. Brisse and others 20 showed that this marker also allowed resolution of two groups within TcIIa corresponding to strains from North America and South America because of unique profiles for each of these groups. This result was also observed here for four additional samples, strengthening the likelihood that there are conserved differences at this locus between North and South American TcIIa strains, and in keeping with some molecular data indicating a significant divergence. 32,35,36 However, caution is needed when drawing such conclusions, as exemplified by the finding that another TcIIa strain, Saimiri3 cl1, has a TcIIb/TcIIe profile. 20 There are additional concerns for the resolution of TcIId because the double band profile that specifies TcIId was not always observed. A single band at this locus is a feature that has also been noted for a small number of other TcIId strains including Sc43 cl1, 10,20 for which the double band profile was detected in this study. This could be a result of differences in experimental conditions or genuine genetic differences between the stocks of the same name in the different laboratories. A drawback of this genotyping method is the requirement to distinguish between bands that are only 5 bp different in size, which can be technically challenging unless appropriate reference strains are used as standards in each analysis.

The mini-exon marker reliably discriminated DTU TcI and a combined group of TcIIb, TcIId, and TcIIe. Typing of DTUs TcIIc and TcIIa was less reliable, in keeping with previous reports. 20 Repeated assays were often needed to confirm TcIIc profiles, and in some cases TcI-like bands were observed, raising the possibility of misclassifications. TcIIa strains gave a number of different profiles, which in some cases would lead to incorrect DTU assignment if this marker was used alone. One reason for the variable TcIIc and TcIIa profiles is the presence of insertions and/or deletions within the target locus, influencing the efficiency of primer binding. 20 Such indels have been characterized in two TcIIc isolates and a TcIIa isolate, 25 but the data presented here suggest that these may not be conserved features within and/or between these sublineages. Furthermore the mini-exon locus exhibits significant secondary structure, 37 which could also adversely affect amplification in some cases. Although the mini-exon assay is clearly inferior to the others tested here in terms of its reliability, direct sequencing of the SL-rRNA locus does have potential for the characterization of intra-DTU diversity, as, for example, shown by a recent study of TcI strains in Colombia. 38

The three PCR-RFLP assays are clearly useful additions to the repertoire of available T. cruzi genotyping protocols. The data presented here are mostly consistent with expectations based on the original study. 27 If each assay is considered separately, there are two cases where unique profiles that are specific to a single DTU were observed. First, the HSP60/EcoRV assay reliably discriminated TcIIc strains, and second, the histone H1/Aat II assay generated TcIIb-specific profiles. The application in combination of the three RFLP markers assessed in this study proved to reliably discriminate all strains into the four non-hybrid DTUs and a fifth combined TcIId/IIe hybrid group, agreeing with the results from the analysis of Westenberger and others 27 of 26 strains. However, exceptions to this general rule occurred in the case of the histone H1/AatII assay for TcIIa(NA) strains and Saimiri3 cl1 and also with the GPI/HhaI assay, again with TcIIa(NA) samples. These discrepancies, caused by point mutations in a relevant restriction site, may or may not reflect a more substantial overall divergence between such North American strains and other members of TcIIa; full sequencing of the target loci will be needed to resolve this question. Overall, these assays were simple to perform and, although they require an extra experimental step and additional reagents compared with the mini-exon or LSU rDNA assays, they seem to be less subject to equivocal results. On the other hand, only LSU rDNA is capable of separating TcIId and TcIIe samples.

None of the individual markers tested here allowed complete DTU resolution, and in any case, reliance on a single marker would be inadvisable because of the consequent loss of resolution and the potential influence of genetic exchange on some lineages. Using a combination of multiple assays, therefore, permits more reliable DTU assignment. Brisse and others 20 proposed a multiple assay system based on a combination of mini-exon, LSU rDNA and 18S rRNA (SSU rDNA) PCR product size polymorphism assays. Although this strategy does permit assignment into each of the six DTUs, the mini-exon assay seems to lack reproducibility in some cases, and several of the assignments depend on the absence, rather than the presence, of bands, which is inadvisable.

The data presented here show that the application of another combination of markers, including PCR-RFLPs, can achieve the same level of resolution with all assignments depending on the presence of specific band sizes. Only the LSU rDNA marker and the mini-exon are able to distinguish TcIIa(NA) from TcI, so one of these assays should be included. The results of this study lead to a strong preference for LSU rDNA because it allows separation of TcIId from TcIIe and is much more reliable for typing of TcIIa and TcIIc. Follow-up with RFLP of HSP60 could resolve all six DTUs. However, each of the RFLPs relies on the presence or absence of either one or two SNPs, which may be affected by mutations in as yet untested strains. It would therefore seem sensible to include a second PCR-RFLP assay in addition to HSP60/EcoRV. The GPI/Hha I assay has two advantages over histone H1/Aat II: first, the absence of non-specific bands, and second, a larger, and therefore more easily visible, smallest digestion product.

In this study, the combination of LSU rDNA PCR with HSP60/EcoRV and GPI/HhaI PCR-RFLPs reliably determined the “correct” DTU for 45 of the 48 cloned isolates. The first exception was that the two TcIIa strains from North America gave TcI/IIc-type RFLP profiles, although they could still be identified by the characteristic 130bp LSU rDNA PCR product. Second, strain Saimiri3 cl1, which has been typed as TcIIa by MLEE and RAPD, 19 had atypical profiles for several of the single-locus genotyping assays used here. The existence of a minority of strains that do not fit comfortably into the DTU concept, should, however, be viewed as an interesting feature of the species rather than an inadequacy of a genotyping system that works most of the time. Complications caused by the existence of such rare isolates or others from as yet unsampled populations are likely to be unavoidable without the application of more markers. Observation of unexpected multilocus genotypes could indicate as yet undiscovered lineages or recombinant strains that warrant further study, for instance by MLST. MLST has not only allowed the identification of recombination in T. cruzi32,33 but also in other eukaryotic pathogens, including Leishmania spp.,39,40 Giardia lamblia,41 Toxoplasma gondii,42 and Candida spp.43,44 Nevertheless, as a tool for simple DTU assignment, our data show that a triple-assay comprising LSU rDNA, HSP60/Eco RV, and GPI/HhaI ( Figure 3 ) represents a good compromise of type-ability of most strains, adequate discriminatory power, reproducibility, and cost, as well as minimal sample material and time requirements.

Further development of multiple locus PCR-RFLP systems will require testing of many more isolates to prove the reliability of each target/enzyme combination. Testing of additional targets is clearly warranted, particularly of ones capable of discriminating between TcIId and TcIIe and any that unequivocally identify TcIIa. Details of other RFLP markers capable of similar levels of resolution were published during the course of this study, including an assay targeted to the GP72 gene using the restriction enzyme TaqI, which does seem to provide discrimination between TcIId and TcIIe. 26 These authors also proved the potential of using PCR-RFLPs to detect diversity of T. cruzi in both clinical and field samples.

In this study aimed at validating genotyping assays, we used a panel of cloned, laboratory cultivated strains. The utility of these assays in practice, however, is subject to the complication of mixed infections, which are well documented in both vectors and mammal hosts, including humans. 4547 Depending on the strains present in such cases, mixed genotype profiles could be observed. For example, mixtures of TcIIb and TcIIc would generate TcIId/IIe profiles for the RFLP assays tested here. It may also be necessary to distinguish between T. cruzi and Trypanosoma rangeli, and this can be done either morphologically, or genetically using an assay that exploits PCR product size differences in the large subunit rRNA gene (LSU rDNA). 48 Furthermore, if there is reason to suspect an isolate belongs to the closely related, bat host-restricted subspecies Trypanosoma cruzi marinkellei, it can be identified by its unique 135-bp band for the LSU rDNA PCR assay. 20

The strains that are used for testing of typing systems need to be carefully considered. Although there are abundant isolates described from domestic transmission cycles, the diversity of T. cruzi in sylvatic settings is less well understood. Indeed, the predominantly sylvatic DTUs TcIIa and TcIIc are often poorly represented in many types of study, including those aimed at characterizing genetic markers. Reducing this sample bias is also particularly important because the success of control strategies targeted at domestic transmission means that the epidemiology of Chagas disease is changing, and adventitious transmission of T. cruzi from sylvatic sources (i.e., TcI, TcIIa, and TcIIc) is seen as increasingly important.2 This is exemplified by cases of acute Chagas disease caused by enzootic transmission in the Brazilian Amazon, which may become increasingly frequent as migration more often brings humans into contact with sylvatic sources of T. cruzi.6 Reliable and reproducible genotyping protocols will aid characterization of new isolates and should contribute to a coordinated research effort across multiple disciplines.

Table 1

Comparison of selected T. cruzi subdivision nomenclature schemes

Table 1
Table 2

Genotype assignment of PCR amplification product sizes (bp) 9,10,20,21,24

Table 2
Table 3

Summary of genotypes

Table 3
Figure 1.
Figure 1.

Example of LSU rDNA and mini-exon PCR product size polymorphism genotyping assay profiles. A, LSU rDNA—Lanes: 1, Rita cl5 (TcIIb); 2, Chaco23 col4 (TcIIb); 3, ARMA18 cl3 (TcIIc); 4, Sc43 cl1 (TcIId); 5, 92–80 cl2 (TcIId); 6, Bug2148 cl1 (TcIId), note absence of 125-bp band; 7, Chaco2 cl3 (TcIId); 8, PAH179 cl5 (TcIId); 9, Para6 cl4 (TcIId); 10, Para4 cl3 (TcIId); 11, Vinch101 cl1 (TcIId); 12, EPV20-1 cl1 (TcIIe); 13, P251 cl7 (TcIIe); 14, VFRA1 cl1 (TcIIe). B, LSU rDNA—Lanes: 1, C8 cl1 (TcI); 2, SAXP18 cl1 (TcI); 3, JR cl4 (TcI); 4, B187 cl10 (TcI); 5, 10R26 (TcIIa); 6, 92122102R [TcIIa(NA)]; 7, CanIII cl1 (TcIIa); 8, StC10R cl1 [TcIIa(NA)]; 9, Saimiri3 cl1 (TcIIa); 10, ERA cl2 (TcIIa); 11, JA2 cl2 (TcIIc); 12, SABP19 cl1 (TcIIc); 13, Vinch101 cl1 (TcIId); 14, LHVA cl4 (TcIIe). Note that comparison of Lanes 8–11 shows the four distinct product sizes: 130, 125, 117, and 110 bp. C, Mini-exon—Lanes: 1, M5631 cl5 (TcIIc); 2, JA2 cl2 (TcIIc); 3, ARMA18 cl3 (TcIIc); 4, 85/847 cl2 (TcIIc); 5, SABP cl1 (TcIIc); 6, VFRA1 cl1 (TcIIe); 7, Chaco2 cl3 (TcIId); 8, Esm cl3 (TcIIb); 9, X10/1 (TcI); 10, B187 cl10 (TcI); 11, JR cl4 (TcI); 12, 92101601P cl1 (TcI); 13, CanIII cl1 (TcIIa); 14, 92122102R [TcIIa(NA)]; 15, Saimiri3 cl1 (TcIIa); 16, X10610 cl5 (TcIIa); 17, ERA cl2 (TcIIa); 18, StC10R cl1 [TcIIa(NA)].

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 81, 6; 10.4269/ajtmh.2009.09-0305

Figure 2.
Figure 2.

Examples of PCR-RFLP genotyping profiles. A, HSP60: digestion products only are shown. Lanes: 1, X10610 cl5 (TcIIa); 2, Saimiri3 cl1 (TcIIa); 3, ERA cl2 (TcIIa); 4, JR cl4 (TcI); 5, 10R26 (TcIIa); 6, StC10R cl1 [TcIIa(NA)]; 7, CanIII cl1 (TcIIa); 8, X10/1 (TcI); 9, 92122102R [TcIIa(NA)]; 10, CJ005/PII (TcI); 11, B187 cl10 (TcI); 12, Chile C22 cl1 (TcI); 13, 92–80 cl2 (TcIId); 14, Rita cl5 (TcIIb); 15, Pot7a cl1 (TcIIb); 16, ARMA18 cl3 (TcIIc); 17, PAH179 cl5 (TcIId); 18, VFRA1 cl1 (TcIIe); 19, SABP19 cl1 (TcIIc); 20, M6241 cl6 (TcIIc); 21, Vinch101 cl1 (TcIId). B, Histone H1: digestion products from unpurified PCR products are shown. Lanes: 1, X10610 cl5 (TcIIa); 2, Saimiri3 cl1 (TcIIa); 3, ERA cl2 (TcIIa); 4, JR cl4 (TcIIc); 5, 10R26 (TcIIa); 6, StC10R cl1 [TcIIa(NA)]; 7, CanIII cl1 (TcIIa); 8, X10/1 (TcI); 9, Pot7b cl5 (TcIIb); 10, Rita cl5 (TcIIb); 11, JA2 cl2 (TcIIc); 12, ARMA13 cl1 (TcIIc); 13, CJ007/PI (TcI); 14, B187 cl10 (TcI); 15, SAXP18 cl1 (TcI); 16, 92122102R [TcIIa(NA)]; 17, SABP19 cl1 (TcIIc); 18, Para4 cl3 (TcIId); 19, CM25 cl2 (TcIIc); 20, PAH179 cl5 (TcIId); 21, Sc43 cl1 (TcIId); 22, Chaco17 col1 (TcIIe); 23, Tu18 cl2 (TcIIb); 24, Chaco23 col4 (TcIIb). C, GPI: each pair of lanes shows undigested PCR product followed by restriction digest products. Lanes: 1, SAXP18 cl1 (TcI); 2, VFRA1 cl1 (TcIIe); 3, StC10R cl1 [TcIIa(NA)]; 4, 10R26 (TcIIa); 5, Pot7a cl1 (TcIIb); 6, Rita cl5 (TcIIb); 7, JA2 cl2 (TcIIc); 8, Para4 cl3 (TcIId); 9, Vinch101 cl1 (TcIId); 10, Chaco9 col15 (TcIIe); 11, CanIII cl1 (TcIIa); 12, ARMA18 cl3 (TcIIc).

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 81, 6; 10.4269/ajtmh.2009.09-0305

Figure 3.
Figure 3.

Recommended triple-assay for discriminating T. cruzi DTUs.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 81, 6; 10.4269/ajtmh.2009.09-0305

*

Address correspondence to Michael D. Lewis, Pathogen Molecular Biology Unit, Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK. E-mail: michael.lewis@lshtm.ac.uk

Authors’ addresses: Michael D. Lewis, Matthew Yeo, Martin S. Llewellyn, and Michael A. Miles, Pathogen Molecular Biology Unit, Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E 7HT, UK, Tel: 44-0-2079-272405, E-mails: michael.lewis@lshtm.ac.uk, matthew.yeo@lshtm.ac.uk, martin.llewellyn@lshtm.ac.uk, and michael.miles@lshtm.ac.uk. Jonathan Ma, Bio-Cancer Treatment International Ltd., Bio-Informatics Centre, 2 Science Park West Avenue, Hong Kong Science Park, Shatin, New Territories, Hong Kong, Tel: 852-2521-1566, E-mail: jonathan.r.ma@ gmail.com. Hernán J. Carrasco, Instituto de Medicina Tropical, Facultad de Medicina, Universidad Central de Venezuela, Caracas, Venezuela, Tel: 58-21260-53546, E-mail: hernan.carrasco@ucv.ve.

Acknowledgments: The authors thank Christian Barnabé, Michael Tibayrenc, and Patricio Diosque who kindly provided T. cruzi strains and Sofia Ocaña and Tapan Bhattacharyya for valuable technical assistance.

Financial support: This work was supported by the BBSRC and the Wellcome Trust. M.S.L. is supported by EC Contract 223034 (ChagasEpiNet). H.J.C. is supported by FONACIT Grant G-2005000827.

REFERENCES

  • 1

    WHO, 2002. World Health Report. Geneva: World Health Organisation.

  • 2

    Schofield CJ, Jannin J, Salvatella R, 2006. The future of Chagas disease control. Trends Parasitol 22 :583–588.

  • 3

    Dias JC, 2007. Southern Cone Initiative for the elimination of domestic populations of Triatoma infestans and the interruption of transfusion Chagas disease: historical aspects, present situation, and perspectives. Mem Inst Oswaldo Cruz 102 (Suppl 1):11–18.

    • Search Google Scholar
    • Export Citation
  • 4

    Feliciangeli MD, Sanchez-Martin MJ, Suarez B, Marrero R, Torrellas A, Bravo A, Medina M, Martinez C, Hernandez M, Duque N, Toyo J, Rangel R, 2007. Risk factors for Trypanosoma cruzi human infection in Barinas State, Venezuela. Am J Trop Med Hyg 76 :915–921.

    • Search Google Scholar
    • Export Citation
  • 5

    Gurtler RE, Kitron U, Cecere MC, Segura EL, Cohen JE, 2007. Sustainable vector control and management of Chagas disease in the Gran Chaco, Argentina. Proc Natl Acad Sci USA 104 :16194–16199.

    • Search Google Scholar
    • Export Citation
  • 6

    Coura JR, Junqueira ACV, Fernandes O, Valente SAS, Miles MA, 2002. Emerging Chagas disease in Amazonian Brazil. Trends Parasitol 18 :171–176.

    • Search Google Scholar
    • Export Citation
  • 7

    Dias JC, Bastos C, Araújo E, Mascarenhas AV, Martins Netto E, Grassi F, Silva M, Tatto E, Mendonça J, Araújo RF, Shikanai-Yasuda MA, Aras R, 2008. Acute Chagas disease outbreak associated with oral transmission. Rev Soc Bras Med Trop 41 :296–300.

    • Search Google Scholar
    • Export Citation
  • 8

    Nóbrega AA, Garcia MH, Tatto E, Obara MT, Costa E, Sobel J, Araujo WN, 2009. Oral transmission of Chagas disease by consumption of açaí palm fruit, Brazil. Emerg Infect Dis 5 :653–655.

    • Search Google Scholar
    • Export Citation
  • 9

    Fernandes O, Souto R, Castro J, Pereira J, Fernandes N, Junqueira A, Naiff R, Barrett T, Degrave W, Zingales B, Campbell D, Coura JR, 1998. Brazilian isolates of Trypanosoma cruzi from humans and triatomines classified into two lineages using mini-exon and ribosomal RNA sequences. Am J Trop Med Hyg 58 :807–811.

    • Search Google Scholar
    • Export Citation
  • 10

    Souto RP, Fernandes O, Macedo AM, Campbell DA, Zingales B, 1996. DNA markers define two major phylogenetic lineages of Trypanosoma cruzi. Mol Biochem Parasitol 83 :141–152.

    • Search Google Scholar
    • Export Citation
  • 11

    Oliveira RP, Broude NE, Macedo AM, Cantor CR, Smith CL, Pena SDJ, 1998. Probing the genetic population structure of Trypanosoma cruzi with polymorphic microsatellites. Proc Natl Acad Sci USA 95 :3776–3780.

    • Search Google Scholar
    • Export Citation
  • 12

    Valadares HMS, Pimenta JR, de Freitas JM, Duffy T, Bartholomeu DC, de Paula Oliveira R, Chiari E, Moreira MCV, Filho GB, Schijman AG, Franco GR, Machado CR, Pena SDJ, Macedo AM, 2008. Genetic profiling of Trypanosoma cruzi directly in infected tissues using nested PCR of polymorphic microsatellites. Int J Parasitol 38 :839–850.

    • Search Google Scholar
    • Export Citation
  • 13

    Llewellyn MS, Miles MA, Carrasco HJ, Lewis MD, Yeo M, Vargas J, Torrico F, Diosque P, Valente V, Valente SA, Gaunt MW, 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 :e1000410.

    • Search Google Scholar
    • Export Citation
  • 14

    Burgos JM, Altcheh J, Bisio M, Duffy T, Valadares HMS, Seidenstein ME, Piccinali R, Freitas JM, Levin MJ, Macchi L, Macedo AM, Freilij H, Schijman AG, 2007. Direct molecular profiling of minicircle signatures and lineages of Trypanosoma cruzi bloodstream populations causing congenital Chagas disease. Int J Parasitol 37 :1319–1327.

    • Search Google Scholar
    • Export Citation
  • 15

    Telleria J, Lafay B, Virreira M, Barnabe C, Tibayrenc M, Svoboda M, 2006. Trypanosoma cruzi: sequence analysis of the variable region of kinetoplast minicircles. Exp Parasitol 114 :279–288.

    • Search Google Scholar
    • Export Citation
  • 16

    Campbell D, Westenberger S, Sturm N, 2004. The determinants of Chagas disease: connecting parasite and host genetics. Curr Mol Med 4 :549–562.

    • Search Google Scholar
    • Export Citation
  • 17

    Momen H, 1999. Taxonomy of Trypanosoma cruzi: a commentary on characterization and nomenclature. Mem Inst Oswaldo Cruz 94 :181–184.

  • 18

    Tibayrenc M, 1998. Genetic epidemiology of parasitic protozoa and other infectious agents: the need for an integrated approach. Int J Parasitol 28 :85–104.

    • Search Google Scholar
    • Export Citation
  • 19

    Brisse S, Barnabe C, Tibayrenc M, 2000. Identification of six Trypanosoma cruzi phylogenetic lineages by random amplified polymorphic DNA and multilocus enzyme electrophoresis. Int J Parasitol 30 :35–44.

    • Search Google Scholar
    • Export Citation
  • 20

    Brisse S, Verhoef J, Tibayrenc M, 2001. Characterisation of large and small subunit rRNA and mini-exon genes further supports the distinction of six Trypanosoma cruzi lineages. Int J Parasitol 31 :1218–1226.

    • Search Google Scholar
    • Export Citation
  • 21

    Kawashita SY, Sanson GFO, Fernandes O, Zingales B, Briones MRS, 2001. Maximum-Likelihood divergence date estimates based on rRNA gene sequences suggest two scenarios of Trypanosoma cruzi intraspecific evolution. Mol Biol Evol 18 :2250–2259.

    • Search Google Scholar
    • Export Citation
  • 22

    Mendonça M, Nehme N, Santos S, Cupolillo E, Vargas N, Junqueira A, Naiff R, Barrett T, Coura J, Zingales B, Fernandes O, 2002. Two main clusters within Trypanosoma cruzi zymodeme 3 are defined by distinct regions of the ribosomal RNA cistron. Parasitology 124 :177–184.

    • Search Google Scholar
    • Export Citation
  • 23

    Ceballos LA, Cardinal MV, Vazquez-Prokopec GM, Lauricella MA, Orozco MM, Cortinas R, Schijman AG, Levin MJ, Kitron U, Gürtler RE, 2006. Long-term reduction of Trypanosoma cruzi infection in sylvatic mammals following deforestation and sustained vector surveillance in northwestern Argentina. Acta Trop 98 :286–296.

    • Search Google Scholar
    • Export Citation
  • 24

    Yeo M, Acosta N, Llewellyn M, Sanchez H, Adamson S, Miles GAJ, Lopez E, Gonzalez N, Patterson JS, Gaunt MW, de Arias AR, Miles MA, 2005. 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 :225–233.

    • Search Google Scholar
    • Export Citation
  • 25

    Fernandes O, Sturm NR, Derre R, Campbell DA, 1998. The mini-exon gene: a genetic marker for zymodeme III of Trypanosoma cruzi. Mol Biochem Parasitol 95 :129–133.

    • Search Google Scholar
    • Export Citation
  • 26

    Rozas M, Doncker SD, Adaui V, Coronado X, Barnabe C, Tibyarenc M, Solari A, Dujardin J-C, 2007. Multilocus polymerase chain reaction restriction fragment-length polymorphism genotyping of Trypanosoma cruzi (Chagas Disease): taxonomic and clinical applications. J Infect Dis 195 :1381–1388.

    • Search Google Scholar
    • Export Citation
  • 27

    Westenberger SJ, Barnabe C, Campbell DA, Sturm NR, 2005. Two hybridization events define the population structure of Trypanosoma cruzi. Genetics 171 :527–543.

    • Search Google Scholar
    • Export Citation
  • 28

    Lewis MD, Llewellyn MS, Gaunt MW, Yeo M, Carrasco HJ, Miles MA, 2009. Flow cytometric analysis and microsatellite genotyping reveal extensive DNA content variation in Trypanosoma cruzi populations and expose contrasts between natural and experimental hybrids. Int J Parasitol 39 :1305–1317.

    • Search Google Scholar
    • Export Citation
  • 29

    Carrasco H, Frame I, Valente S, Miles M, 1996. Genetic exchange as a possible source of genomic diversity in sylvatic populations of Trypanosoma cruzi. Am J Trop Med Hyg 54 :418–424.

    • Search Google Scholar
    • Export Citation
  • 30

    Sturm NR, Vargas NS, Westenberger SJ, Zingales B, Campbell DA, 2003. Evidence for multiple hybrid groups in Trypanosoma cruzi. Int J Parasitol 33 :269–279.

    • Search Google Scholar
    • Export Citation
  • 31

    Gaunt MW, Yeo M, Frame IA, Stothard JR, Carrasco HJ, Taylor MC, Mena SS, Veazey P, Miles GAJ, Acosta N, de Arias AR, Miles MA, 2003. Mechanism of genetic exchange in American trypanosomes. Nature 421 :936–939.

    • Search Google Scholar
    • Export Citation
  • 32

    Brisse S, Henriksson J, Barnabe C, Douzery EJP, Berkvens D, Serrano M, De Carvalho MRC, Buck GA, Dujardin J-C, Tibayrenc M, 2003. Evidence for genetic exchange and hybridization in Trypanosoma cruzi based on nucleotide sequences and molecular karyotype. Infect Genet Evol 2 :173–183.

    • Search Google Scholar
    • Export Citation
  • 33

    Machado CA, Ayala FJ, 2001. Nucleotide sequences provide evidence of genetic exchange among distantly related lineages of Trypanosoma cruzi. Proc Natl Acad Sci USA 98 :7396–7401.

    • Search Google Scholar
    • Export Citation
  • 34

    de Freitas JM, Augusto-Pinto L, Pimenta JR, Bastos-Rodrigues L, Gonçalves VF, Teixeira SMR, Chiari E, Junqueira AnCV, Fernandes O, Macedo AM, Machado CR, Pena SDJ, 2006. Ancestral genomes, sex, and the population structure of Trypanosoma cruzi. PLoS Pathog 2 :24.

    • Search Google Scholar
    • Export Citation
  • 35

    Barnabé C, Yaeger R, Pung O, Tibayrenc M, 2001. Trypanosoma cruzi: a considerable phylogenetic divergence indicates that the agent of Chagas disease is indigenous to the native fauna of the United States. Exp Parasitol 99 :73–79.

    • Search Google Scholar
    • Export Citation
  • 36

    Marcili A, Lima L, Valente VC, Valente SA, Batista JS, Junqueira AC, Souza AI, da Rosa JA, Campaner M, Lewis MD, Llewellyn MS, Miles MA, Teixeira MM, 2009. Comparative phylogeography of Trypanosoma cruzi TCIIc: new hosts, association with terrestrial ecotopes, and spatial clustering. Infect Genet Evol (In press).

    • Search Google Scholar
    • Export Citation
  • 37

    Thomas S, Westenberger SJ, Campbell DA, Sturm NR, 2005. Intragenomic spliced leader RNA array analysis of kineto-plastids reveals unexpected transcribed region diversity in Trypanosoma cruzi. Gene 352 :100–108.

    • Search Google Scholar
    • Export Citation
  • 38

    Herrera C, Bargues MD, Fajardo A, Montilla M, Triana O, Vallejo GA, Guhl F, 2007. Identifying four Trypanosoma cruzi I isolate haplotypes from different geographic regions in Colombia. Infect Genet Evol 7 :535–539.

    • Search Google Scholar
    • Export Citation
  • 39

    Mauricio IL, Yeo M, Baghaei M, Doto D, Pratlong F, Zemanova E, Dedet J-P, Lukes J, Miles MA, 2006. Towards multilocus sequence typing of the Leishmania donovani complex: resolving genotypes and haplotypes for five polymorphic metabolic enzymes (ASAT, GPI, NH1, NH2, PGD). Int J Parasitol 36 :757–769.

    • Search Google Scholar
    • Export Citation
  • 40

    Ravel C, Cortes S, Pratlong F, Morio F, Dedet J-P, Campino L, 2006. First report of genetic hybrids between two very divergent Leishmania species: Leishmania infantum and Leishmania major. Int J Parasitol 36 :1383–1388.

    • Search Google Scholar
    • Export Citation
  • 41

    Cooper MA, Adam RD, Worobey M, Sterling CR, 2007. Population genetics provides evidence for recombination in Giardia. Curr Biol 17 :1984–1988.

    • Search Google Scholar
    • Export Citation
  • 42

    Boyle JP, Rajasekar B, Saeij JPJ, Ajioka JW, Berriman M, Paulsen I, Roos DS, Sibley LD, White MW, Boothroyd JC, 2006. Just one cross appears capable of dramatically altering the population biology of a eukaryotic pathogen like Toxoplasma gondii. Proc Natl Acad Sci USA 103 :10514–10519.

    • Search Google Scholar
    • Export Citation
  • 43

    Dodgson AR, Pujol C, Pfaller MA, Denning DW, Soll DR, 2005. Evidence for recombination in Candida glabrata. Fungal Genet Biol 42 :233–243.

    • Search Google Scholar
    • Export Citation
  • 44

    Tavanti A, Gow NAR, Maiden MCJ, Odds FC, Shaw DJ, 2004. Genetic evidence for recombination in Candida albicans based on haplotype analysis. Fungal Genet Biol 41 :553–562.

    • Search Google Scholar
    • Export Citation
  • 45

    Bosseno M-F, Telleria J, Vargas F, Yaksic N, Noireau F, Morin A, Brenière SF, 1996. Trypanosoma cruzi: study of the distribution of two widespread clonal genotypes in Bolivian Triatoma infestans vectors shows a high frequency of mixed infections. Exp Parasitol 83 :275–282.

    • Search Google Scholar
    • Export Citation
  • 46

    Cardinal MV, Lauricella MA, Ceballos LA, Lanati L, Marcet PL, Levin MJ, Kitron U, Gürtler RE, Schijman AG, 2008. Molecular epidemiology of domestic and sylvatic Trypanosoma cruzi infection in rural northwestern Argentina. Int J Parasitol 38 :1533–1543.

    • Search Google Scholar
    • Export Citation
  • 47

    Yeo M, Lewis MD, Carrasco HJ, Acosta N, Llewellyn M, da Silva Valente SA, de Costa Valente V, de Arias AR, Miles MA, 2007. Resolution of multiclonal infections of Trypanosoma cruzi from naturally infected triatomine bugs and from experimentally infected mice by direct plating on a sensitive solid medium. Int J Parasitol 37 :111–120.

    • Search Google Scholar
    • Export Citation
  • 48

    Souto RP, Vargas N, Zingales B, 1999. Trypanosoma rangeli: discrimination from Trypanosoma cruzi based on a variable domain from the large subunit ribosomal RNA gene. Exp Parasitol 91 :306–314.

    • Search Google Scholar
    • Export Citation
  • 49

    Miles MA, Yeo M, Gaunt MW, 2003. Genetic diversity of Trypanosoma cruzi and the epidemiology of Chagas disease. Kelly JM, ed. Molecular Mechanisms of Pathogenesis in Chagas Disease. New York: Kluwer Academic, 1–15.

  • 50

    Tibayrenc M, Ayala F, 1988. Isozyme variability of Trypanosoma cruzi, the agent of Chagas’ disease: genetical, taxonomical and epidemiological significance. Evolution 42 :277–292.

    • Search Google Scholar
    • Export Citation
  • 51

    Anonymous, 1999. Recommendations from a satellite meeting. Mem Inst Oswaldo Cruz 94 :429–432.

  • 52

    Roellig DM, Brown EL, Barnabe C, Tibayrenc M, Steurer FJ, Yabsley MJ, 2008. Molecular typing of Trypanosoma cruzi isolates, United States. Emerg Infect Dis 14 :1123–1125.

    • Search Google Scholar
    • Export Citation
Save