Quantitative Real-Time PCR for Trypanosoma cruzi Infection Diagnosis and Treatment Response Monitoring of Patients with Chagas Disease

Abstract

The development of accurate diagnostic tools and surrogate markers of parasitological response to treatment are priorities in Chagas disease (CD) research. For years, the detection of Trypanosoma cruzi DNA by polymerase chain reaction (PCR) has proved to be useful in some clinical scenarios such as acute CD, including cases of vertical transmission and oral outbreaks, CD reactivation in immunosuppressed patients, and post-treatment follow-up. In that sense, the implementation of quantitative real-time PCR (qPCR) assays was an important step in the development of more reliable tools for T. cruzi infection diagnosis and treatment response monitoring. In 2011, a multicenter study addressed the analytical validation and clinical evaluation of two multiplex qPCR strategies for the quantification of T. cruzi DNA in blood samples. This chapter describes the most-used protocol to detect and quantify parasite load in human blood samples, including dried blood spots on filter paper, by T. cruzi satellite DNA multiplex qPCR with endogenous and exogenous internal amplification controls, and discusses some aspects to consider during planning and executing these procedures.