Standardization of Trypanosoma cruzi DNA extraction and purification protocol from samples collected on Whatman 903 filter paper to Chagas disease diagnosis

Abstract

Chagas disease (CD) caused by the parasite Trypanosoma cruzi, belongs to the so-called neglected diseases group. In Argentina about 1,500 children are born with congenital Chagas disease per year. The diagnosis of CD in the newborn relies on the ability to detect parasites in the blood by microscopic observation, as the serological tests are ruled out because of the presence of maternal antibodies. CD treatment is more effective during the acute phase of infection. Early diagnosis and treatment of the disease is thus very important. The Argentinian National Program for early detection of metabolic diseases uses Whatman903 filter paper for blood sampling. This type of sample collection presents many advantages as the use of low blood volumes, minimal biological risk, and easy storage and transportation. The objective of the study was to evaluate the conservation efficiency of blood samples on filter paper in order to access good sensitivity on qPCR results for the detection of T. cruzi. To standardize the procedure, negative samples of blood were infected artificially with serial dilutions of trypomastigotes forms of T. cruzi from the TcVI strain obtained by cell culture in Vero cells. Concentrations between 50000 and 5 parasites/mL were prepared and loaded in filter paper for analysis. DNA extraction was conducted by the QIAamp DNA Mini Kit from QIAGEN. For qPCR, a method based on TaqMan technology was used, with a multiplex reaction for quantification of T. cruzi satellite DNA and an internal amplification control (IAC). The detection limit found from our results was 400 parasites/mL, demonstrating that this method could be a reliable option for the diagnosis of congenital CD by the detection of T. cruzi in blood collected in filter paper.