Diagnóstico parasitológico e molecular da doença de Chagas humana e tipagem do Trypanosoma cruzi recém-isolado de pacientes portadores de diferentes formas clínicas

Abstract

This study evaluated two diagnostic methods for Chagas disease, such as blood culture and polymerase chain reaction (PCR) as well as genetic profile of Trypanosoma cruzi populations freshly isolated from 91 untreated chronic chagasic patients with defined clinical forms, indeterminate (n=23) and cardiac (n=68). Total positivity of blood culture was 54.95%, and 60.29% of cardiac patients and 39.13% of patients with indeterminate clinical form were positive. T. cruzi was detected in 70.00% of blood cultures between 30-60 days, and 56.00% of patients had one or two positive tubes. T. cruzi kDNA was detected by PCR in 98.90% of patients, in relation to clinical form; the positivity was 100.00% in cardiac patients and 95.65% of patients with indeterminate clinical form. The association between increased positivity in 43.96%, since 54.95% of patients were positive by both PCR and blood culture and 1.09% were negative by both methods. The association of these methods increased positivity in 43.96%, since 54.95% of patients were positive by both PCR and blood culture, and 1.09% were negative by both methods. The genetic variability was analyzed in 36 isolates of T. cruzi, 29 chagasic patients with heart disease and seven patients with indeterminate form using molecular markers: divergent domain D7 24S rDNA gene, mitochondrial gene cytochrome oxidase subunit II (CO II) and intergenic region of genes miniexon (SL-IRac), and 97.22% of T. cruzi isolates were related to rDNA 1 and mitochondrial haplogroup C associated with DTU II. However, an isolate from a patient with indeterminate clinical form was associated to DTU III or V by rDNA 24s marker and DTU I with CO II. The analysis with SL-IRac confirmed that this isolate corresponds to DTU I. The data demonstrated that blood culture and PCR were efficient with just a blood sample and, when associated increased sensitivity in the diagnosis of chronic Chagas disease. PCR is more sensitive for the diagnosis, since it was able to detect parasite kDNA in blood culture negative patients. The genetic profile of the isolates of T. cruzi showed that it was not possible to establish an association with the clinical forms of chagasic patients.