Parasites by QBC, for blood smear, for indirect xenodiagnosis, for blood culture and for serology for the detection of total IgM and anti-T. cruzi IgG antibodies by indirect immunofluorescence assay (indirect IFA) and indirect hemagglutination assay (IHA). All of the above assays were performed pre-treatment (0 days) and repeated 35 (67) and 68 (66) days after the onset of treatment or during the immediate post-treatment Epigenetics period. With the exception of direct sampling of T. cruzi, the other tests were repeated every 6 Autophagy months after the end of treatment while the patient remained seropositive, with the last collection performed in 2005 (follow-up survey). Monitoring was performed individually with monthly assessments during treatment and every 6 months thereafter. The last assessment was conducted in 2005. Indirect xenodiagnoses. was performed by abdominal compression and collection of sample feces. Samples were placed between a slide and cover slip and observed with an optical microscope (OM). These observations were taken between 30 and 45 days, and again at 60 days after insects feeding. Results were expressed as positive or negative. Blood culture: 1 ml blood sample collected from each patient was distributed into five tubes containing Hoff culture medium, prepared as described by Abramo et al. (1980) [11]. Treatment failure was recorded if a positive result from one of those parasitological exams was obtained at any time after the completion of treatment (6866 days).Radiological Evaluation of the Digestive TractRadiological contrast exams of the esophagus and colon were performed on 33 adults who had acute infection for more than four years at the Radiology department of Santa Casa de Misericordia do Para Hospital Foundation. For colons radiology ??patients received information about the procedures and underwent panoramic radiography of the abdomen in an anteriorposterior (AP) position. The contrast agent contained 350 ml of Telebrix (barium sulfate) and 50 ml of saline solution in a final volume of 400 ml. Radiological procedures were performed as follows: a) overview of the abdomen in the posterior-anterior (PA) position; b) right side anterior-posterior position; and c) lateral rectum. After evacuation, patients returned to the examination table for an AP radiograph of 23148522 the abdomen. For contrast examination of the esophagus, a barium sulfate-based suspension was ingested. With patients in a standing position, three positions were recorded by X-ray: oblique, to observe swallowing disorders; frontal, to observe the gastric mucosa; and in profile, to observe the thoracic esophagus.Polymerase Chain Reaction (PCR)We collected 10 ml of blood from 72 randomly chosen patients among those from follow-up. The collected blood was placed directly into tubes containing a solution of guanidine HCl/0.2 M EDTA and stored at 7uC. Tubes containing blood-guanidineEDTA were partially immersed in water and boiled for 15 minutes to linearize and release minicircles from the concatenated K-DNA network. A 100-ml aliquot was used for the preparation of DNA. Extraction procedures were performed in duplicate. After deproteinization using phenol-chloroform (1:1) and chloroformsaturated water, 10 sodium acetate and two volumes of ethanol were added. The mixture was incubated for 15 min on ice and then centrifuged at 12,000 rpm for 15 min. The supernatant was discarded, and the pellet was dried on a hot plate (Multi-block) at 70uC for 5 min. The pellet was resus.Parasites by QBC, for blood smear, for indirect xenodiagnosis, for blood culture and for serology for the detection of total IgM and anti-T. cruzi IgG antibodies by indirect immunofluorescence assay (indirect IFA) and indirect hemagglutination assay (IHA). All of the above assays were performed pre-treatment (0 days) and repeated 35 (67) and 68 (66) days after the onset of treatment or during the immediate post-treatment period. With the exception of direct sampling of T. cruzi, the other tests were repeated every 6 months after the end of treatment while the patient remained seropositive, with the last collection performed in 2005 (follow-up survey). Monitoring was performed individually with monthly assessments during treatment and every 6 months thereafter. The last assessment was conducted in 2005. Indirect xenodiagnoses. was performed by abdominal compression and collection of sample feces. Samples were placed between a slide and cover slip and observed with an optical microscope (OM). These observations were taken between 30 and 45 days, and again at 60 days after insects feeding. Results were expressed as positive or negative. Blood culture: 1 ml blood sample collected from each patient was distributed into five tubes containing Hoff culture medium, prepared as described by Abramo et al. (1980) [11]. Treatment failure was recorded if a positive result from one of those parasitological exams was obtained at any time after the completion of treatment (6866 days).Radiological Evaluation of the Digestive TractRadiological contrast exams of the esophagus and colon were performed on 33 adults who had acute infection for more than four years at the Radiology department of Santa Casa de Misericordia do Para Hospital Foundation. For colons radiology ??patients received information about the procedures and underwent panoramic radiography of the abdomen in an anteriorposterior (AP) position. The contrast agent contained 350 ml of Telebrix (barium sulfate) and 50 ml of saline solution in a final volume of 400 ml. Radiological procedures were performed as follows: a) overview of the abdomen in the posterior-anterior (PA) position; b) right side anterior-posterior position; and c) lateral rectum. After evacuation, patients returned to the examination table for an AP radiograph of 23148522 the abdomen. For contrast examination of the esophagus, a barium sulfate-based suspension was ingested. With patients in a standing position, three positions were recorded by X-ray: oblique, to observe swallowing disorders; frontal, to observe the gastric mucosa; and in profile, to observe the thoracic esophagus.Polymerase Chain Reaction (PCR)We collected 10 ml of blood from 72 randomly chosen patients among those from follow-up. The collected blood was placed directly into tubes containing a solution of guanidine HCl/0.2 M EDTA and stored at 7uC. Tubes containing blood-guanidineEDTA were partially immersed in water and boiled for 15 minutes to linearize and release minicircles from the concatenated K-DNA network. A 100-ml aliquot was used for the preparation of DNA. Extraction procedures were performed in duplicate. After deproteinization using phenol-chloroform (1:1) and chloroformsaturated water, 10 sodium acetate and two volumes of ethanol were added. The mixture was incubated for 15 min on ice and then centrifuged at 12,000 rpm for 15 min. The supernatant was discarded, and the pellet was dried on a hot plate (Multi-block) at 70uC for 5 min. The pellet was resus.
