Real-time PCR strategy for parasite quantification in blood and tissue samples of experimental Trypanosoma cruzi infection

Abstract

The lack of an accurate diagnosis has been a serious obstacle to the advancement of the anti-Trypanosoma cruzi chemotherapy and long-term infection can result in different health risks to human. PCRs are alternative methods, more sensitive than conventional parasitological techniques, which due to their low sensitivities are considered unsuitable for these purposes. The aim of this study was to investigate a sensitive diagnostic strategy to quantify blood and cardiac tissues parasites based on real-time PCR tools during acute and chronic phases of murine Chagas disease, as well as to monitor the evolution of infection in those mice under specific treatment. In parallel, fresh blood examination, immunological analysis and quantification of cardiac inflammation were also performed to confront and improve real-time PCR data. Similar profiles of parasitemia curves were observed in both quantification techniques during the acute phase of the infection. In contrast, parasites could be quantified only by real-time PCR at 60 and 120 days of infection. In cardiac tissue, real-time PCR detected T. cruzi DNA in 100% of infected mice, and using this tool a significant Pearson correlation between parasite load in peripheral blood and in cardiac tissue during acute and chronic phases was observed. Levels of serum CCL2, CCL5 and nitric oxide were coincident with parasite load but focal and diffuse mononuclear infiltrates was observed, even with significant (p<0.05) reduction of parasitism after 60 days of infection. Later, this methodology was used to monitor the evolution of infection in animals treated with itraconazole (Itz). Itz-treatment induced a reduction of parasite load in both blood and cardiac muscle at the treatment period, but after the end of chemotherapy an increase of parasitism was detected. Interestingly, inflammatory mediators levels and heart inflammation intensity had similar evolution to the parasite load, in the group of animals treated. Taken together, our data show that real-time PCR strategy used was suitable for studies of murine T. cruzi infection and may prove useful in investigations involving experimental chemotherapy of the disease and the benefits of treatment in relation to parasitism and inflammatory response.

Altered Cardiomyocyte Function and Trypanosoma cruzi Persistence in Chagas Disease

“…It is interesting to note, however, that most of the previous studies detected T. cruzi in cardiac tissue and not in isolated cardiomyocytes. 30,31 Here, we detected the presence of T. cruzi in isolated cardiomyocytes, which strongly indicates that parasites are present in cardiomyocyte cells. However, we cannot completely rule out the presence of infected cells isolated along with cardiomyocytes during the enzymatic dissociation of the heart.…”

Dengue Patients with Early Hemorrhagic Manifestations Lose Coordinate Expression of the Anti-Inflammatory Cytokine IL-10 with the Inflammatory Cytokines IL-6 and IL-8

“…30 The RNase P internal control was used as an important strategy for accurate quantification of the RNA viral load, allowing us to monitor possible false-negative results (due to RNA degradation) and to correct for variations in the amounts of the initial sample (due to different RNA recovery), thereby allowing for PCR data normalization. 29 Standard curves were generated by 10-fold serial dilutions of transcribed RNAs ranging from 10 7 to 10 3 copies/μL and used in a one-step quantitative real-time PCR (qRT-PCR). 30 Quantitation was performed using the StepOnePlus System The cycling program consisted of a reverse transcription step at 50°C for 30 minutes and initial denaturation at 95°C for 2 minutes, followed by 40 cycles at 95°C for 15 seconds and 60°C for 1 minute.…”

Curcumin Enhances the Anti-Trypanosoma cruzi Activity of Benznidazole-Based Chemotherapy in Acute Experimental Chagas Disease

“…Animals that were positive in any test of infection recrudescence and those in which parasite DNA was detected were considered not cured (26). Extraction of total genomic DNA from cardiac tissue was performed using a commercial kit (Genomic DNA purification kit; Promega) according to the method of Caldas et al (29). DNA concentrations were adjusted to 25 ng/l (GeneQuant; Pharmacia Biotech, Piscataway, NJ, USA).…”