Molecular Genotyping of Trypanosoma cruzi for Lineage Assignment and Population Genetics

  • Louisa A. Messenger
  • Matthew Yeo
  • Michael D. Lewis
  • Martin S. Llewellyn
  • Michael A. MilesEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1201)


Trypanosoma cruzi, the etiological agent of Chagas disease, remains a major public health problem in Latin America. Infection with T. cruzi is lifelong and can lead to a spectrum of pathological sequelae ranging from subclinical to lethal cardiac and/or gastrointestinal complications. Isolates of T. cruzi can be assigned to six genetic lineages or discrete typing units (DTUs), which are broadly associated with disparate ecologies, transmission cycles, and geographical distributions. This extensive genetic diversity is also believed to contribute to the clinical variation observed among chagasic patients. Unravelling the population structure of T. cruzi is fundamental to understanding Chagas disease epidemiology, developing control strategies, and resolving the relationship between parasite genotype and clinical prognosis.

To date, no single, widely validated, genetic target allows unequivocal resolution to DTU-level. In this chapter we present standardized methods for strain DTU assignment using PCR-restriction fragment length polymorphism analysis (PCR-RFLP) and nuclear multilocus sequence typing (MLST). PCR-RFLPs have the advantages of simplicity and reproducibility, requiring limited expertise and few laboratory consumables. MLST data are more laborious to generate but more informative; DNA sequences are readily transferable between research groups and amenable to recombination detection and intra-lineage analyses. We also recommend a mitochondrial (maxicircle) MLST scheme and a panel of 28 microsatellite loci for higher resolution population genetics studies.

Due to the scarcity of T. cruzi in blood and tissue, all of these genotyping techniques have limited sensitivity when applied directly to clinical or biological specimens, particularly when targets are single (MLST) or low copy number (PCR-RFLPs). We therefore describe essential protocols to isolate parasites, derive biological clones, and extract T. cruzi genomic DNA from field and clinical samples.

Key words

Trypanosoma cruzi PCR Genotyping Phylogenetics Microsatellites MLST RFLP Mitochondria Sequencing 



Research detailed in this chapter was funded by support from the Wellcome Trust, the BBSRC and the European Union Seventh Framework Programme grant 223034 (“ChagasEpiNet”).


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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Louisa A. Messenger
    • 1
  • Matthew Yeo
    • 2
  • Michael D. Lewis
    • 2
  • Martin S. Llewellyn
    • 3
  • Michael A. Miles
    • 3
    Email author
  1. 1.London School of Hygiene and Tropical MedicineLondonUK
  2. 2.London School of Hygiene and Tropical MedicineLondonUK
  3. 3.London School of Hygiene and Tropical MedicineLondonUK

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