A Simple Strain Typing Assay for Trypanosoma cruzi: Discrimination of Major Evolutionary Lineages from a Single Amplification Product

Abstract

Background

Trypanosoma cruzi is the causative agent of Chagas' Disease. The parasite has a complex population structure, with six major evolutionary lineages, some of which have apparently resulted from ancestral hybridization events. Because there are important biological differences between these lineages, strain typing methods are essential to study the T. cruzi species. Currently, there are a number of typing methods available for T. cruzi, each with its own advantages and disadvantages. However, most of these methods are based on the amplification of a variable number of loci.

Methodology/Principal Findings

We present a simple typing assay for T. cruzi, based on the amplification of a single polymorphic locus: the TcSC5D gene. When analyzing sequences from this gene (a putative lathosterol/episterol oxidase) we observed a number of interesting polymorphic sites, including 1 tetra-allelic, and a number of informative tri- and bi-allelic SNPs. Furthermore, some of these SNPs were located within the recognition sequences of two commercially available restriction enzymes. A double digestion with these enzymes generates a unique restriction pattern that allows a simple classification of strains in six major groups, corresponding to DTUs TcI–TcIV, the recently proposed Tcbat lineage, and TcV/TcVI (as a group). Direct sequencing of the amplicon allows the classification of strains into seven groups, including the six currently recognized evolutionary lineages, by analyzing only a few discriminant polymorphic sites.

Conclusions/Significance

Based on these findings we propose a simple typing assay for T. cruzi that requires a single PCR amplification followed either by restriction fragment length polymorphism analysis, or direct sequencing. In the panel of strains tested, the sequencing-based method displays equivalent inter-lineage resolution to recent multi- locus sequence typing assays. Due to their simplicity and low cost, the proposed assays represent a good alternative to rapidly screen strain collections, providing the cornerstone for the development of robust typing strategies.

Author Summary

Trypanosoma cruzi, the causative agent of Chagas Disease, infects approximately 8 million people in the Americas, with 200,000 new cases reported anually. The disease, in its chronic stage, has different manifestations: mega-colon, mega-esophagus, and cardiomyopathy, among others. The fact that infections by the same species cause these different clinical outcomes is believed to be determined, at least in part, by the genetic background of the parasite (infection by different strains). By analyzing a number of molecular markers, the population of the parasite has been divided into seven major evolutionary lineages, which evolve mostly independently, by clonal expansion with infrequent exchange of genetic material. Accurate classification of different strains and isolates into their corresponding evolutionary lineages is therefore essential to obtain a good map of biological, biochemical and ecoepidemiological features for the whole species. The current methods available to type T. cruzi stocks are either laborious and costly (requiring the amplification and sequencing of a variable number of genes or gene fragments), or limited in resolution. In this work we describe a number of key discriminant sites in a gene encoding a putative enzyme from the sterol pathway of the parasite, which were exploited to design a couple of alternative typing assays. Using these key discriminant sites, we can classify any T. cruzi stock into either six or seven evolutionary lineages using only one gene fragment, and in a matter of hours (depending on the assay used). To our knowledge, the proposed assays are the first typing assays that can discriminate T. cruzi stocks with such speed and low cost.