title: 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.
authors.affiliation: Núcleo de Pesquisas em Ciências Biológicas, Universidade Federal de Ouro Preto, Campus universitário, Morro do Cruzeiro, Ouro Preto, Minas Gerais, Brazil.
authors.forename: Sérgio
authors.initials: S
authors.lastname: Caldas
authors.forename: Ivo Santana
authors.initials: IS
authors.lastname: Caldas
authors.forename: Lívia de Figueiredo
authors.initials: Lde F
authors.lastname: Diniz
authors.forename: Wanderson Geraldo de
authors.initials: WG
authors.lastname: Lima
authors.forename: Riva de Paula
authors.initials: Rde P
authors.lastname: Oliveira
authors.forename: Alzira Batista
authors.initials: AB
authors.lastname: Cecílio
authors.forename: Isabela
authors.initials: I
authors.lastname: Ribeiro
authors.forename: André
authors.initials: A
authors.lastname: Talvani
authors.forename: Maria Terezinha
authors.initials: MT
authors.lastname: Bahia
mesh_terms: D000818:Animals; D000981:Antiprotozoal Agents / Q000008:administration & dosage; D001769:Blood / Q000469:parasitology; D014355:Chagas Disease / Q000188:drug therapy / Q000469:parasitology*; D018932:Chemokine CCL2 / Q000097:blood; D018946:Chemokine CCL5 / Q000097:blood; D004195:Disease Models, Animal; D005260:Female; D006321:Heart / Q000469:parasitology; D017964:Itraconazole / Q000008:administration & dosage; D051379:Mice; D009569:Nitric Oxide / Q000097:blood; D059208:Parasite Load / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D014349:Trypanosoma cruzi / Q000302:isolation & purification*
chemical_list: D000981:Antiprotozoal Agents; C491768:Ccl2 protein, mouse; C503460:Ccl5 protein, mouse; D018932:Chemokine CCL2; D018946:Chemokine CCL5; D017964:Itraconazole; D009569:Nitric Oxide