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Fifty individuals with chronic chagasic cardiopathy were randomly selected group A and 50 without cardiopathy group B. Echo-Doppler was performed on the patients of group A to eliminate other important known causes of cardiopathy, hypertension, valve disease, atherosclerosis, Phone sex in illapel, and congenital malformations. Informed consent from patients was given in written form. In each determination, positive and negative controls from chagasic and non-chagasic individuals were included. The optical density corresponding to the cutoff value was determined by the average of the negative control plus 0.

XD was applied using two cylindrical wooden boxes each containing seven uninfected third or fourth instar nymphs of T. The criteria to determine a negative XD was the absence of mobile forms of T. Electrocardiographic tracing The patients were evaluated by a twelve-lead electrocardiogram. Each electrocardiographic trace included the following parameters: The final interpretation of this test data was performed by a specialist cardiologist following the double blind protocol recommended by the World Health Organization; the investigator analyzing the ECG traces was unaware of the status of the patients Maguire et al. The following parameters were measured with bi-dimensional M mode: The Doppler measurements allowed the determination of the status of the different valves and the presence of reflux.

The final interpretation of this test was performed by a cardiologist specialist in echography. The final concentrations of the reagents were as follows: Each experiment included two negative PCR controls: As positive control purified DNA of T. To obtain a standard curve to perform the quantification, we used a stock of epimastigote forms of T. The DNA was diluted 1: The standard curve of T. The controls used for qPCR-T. The control mixture and water control are useful to evaluate contamination in the preparation of the mixture reaction or unspecific qPCR reactions.

Each point of the standard curve was performed in triplicate. The samples and controls were included in duplicate. X12 primers were designed by N. Jullien using the AmplifX v. The thermals profiles of qPCR-T. In 21 of them, the concentration of PCR products was below the minimum required for hybridization tests. These fragments were further digested with restriction endonucleases Sau96I and ScaI Fermentas, Ontario, Canada to obtain a bp band and remove all sequences from the minicircle constant region. This method has been validated by hybridization with the probes constructed by PCR amplification of T.

The hybridization profiles were analyzed to compare the intensity of the ethidium bromide-stained bands in each membrane with the presence of the radioactive bands obtained with each probe. The specificity of the probes was tested in hybridization controls against DNA of the probes themselves. The profiles of radioactive hybridization signals with multiple probes are indicative of mixed infections with several genotypes of T. The description of the data was performed by tables, arithmetic mean, mode, standard deviation, and amplitude of the variable.

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