A new Trypanosoma cruzi genotyping method enables high resolution evolutionary analyses

Abstract

BACKGROUND Trypanosoma cruzi is an important human pathogen in Latin America with nearly seven million people infected. It has a large degree of genetic diversity, classified into six discrete typing units (DTUs), which probably influences its physiological behavior and clinical manifestations. Several genotyping methods are available, with distinct performance on easiness, cost, resolution and applicability; no method excels in all parameters. OBJECTIVES AND METHODS To devise a molecular method for T. cruzi genotyping, based on polymerase chain reaction (PCR) amplification of a single target with multiple copies in the nuclear genome by large scale sequencing. We have applied this method to 29 T. cruzi isolates, comprising all described DTUs. FINDINGS We were able to classify all samples into sub DTU level with high robustness. Evolutionary relationship between DTUs were ascertained, suggesting that TcIII and TcIV DTUs are non-hybrid, and DTU IV is more similar to the common ancestral. CONCLUSION As the TS-LSS method is based on a single PCR reaction, comprising several copies of the target, it is probably useful for clinical samples, when the amount of DNA is a limiting factor. As large scale sequencing systems become more common, the TS-LSS method can be increasingly applied for T. cruzi genotyping.

Comparative Assessment of Two Commercial Real-Time PCR Assays for the Diagnosis of Trypanosoma cruzi DNA in Serum

“…Both real-time PCR assays correctly identified DNA of the six discrete typing units (DTUs) of T. cruzi described in the literature [19] as well, suggesting high conservation of the sequence targets and thus applicability in different geographic regions. The DTU assessment was performed with highly concentrated pathogen DNA, as reflected by very low measured Ct values, although absolute quantification data from the extracted culture materials were not available.…”

“…The mean age ± standard deviation was 22.3 ± 18.3 years, and the median age (interquartile range) was 15 (8.34) years (Table 1). In addition to the patients' samples, nucleic acid extractions of the six discrete typing units (DTUs) of T. cruzi, as described recently [19], were included in the comparative realtime PCR assessment. The DTU DNA was a donation from the University del Rosario, Bogota, Colombia, and had been extracted from cultured epimastigote cells, as described by the donator elsewhere [4,15,20].…”