We tested two Trypanosoma cruzi recombinant antigens in a diagnostic test for Chagas' disease. These antigens were a cytoplasmic repetitive antigen (CRA) and a flagellar repetitive antigen (FRA). The results indicate that the recombinant antigens give better results when used in combination than when used separately, and that the removal of the beta-galactosidase portion of the recombinant fusion proteins increases the specificity of the diagnostic test for Chagas' disease. In addition, a direct enzyme-linked immunosorbent assay (ELISA), which involves the use of peroxidase-labeled antigens to detect the immune-complexes, was developed and compared with a conventional ELISA. The results indicate that the recombinant (CRA+FRA) ELISA is better than the conventional ELISA in the diagnosis of Chagas' disease, providing 100% specificity and sensitivity in all sera tested to date. The recombinant ELISA was compared with conventional serologic tests (hemagglutination and immunofluorescence) for Chagas' disease diagnosis, and the results show that the recombinant ELISA does not give rise to false-positive results that are observed with the two other tests. The use of the recombinant ELISA should be useful in the prevention of transmission of Chagas' disease by blood transfusions.

No abstract

Leprosy is a chronic dermato-neurological disease caused by Mycobacterium leprae, an obligate intracellular bacterium. Timely detection is a challenge in leprosy diagnosis, relying on clinical examination and trained health professionals. Furthermore, adequate care and transmission control depend on early and reliable pathogen detection. Here, we describe a qPCR test for routine diagnosis of leprosy-suspected patients. The reaction simultaneously amplifies two specific Mycobacterium leprae targets (16S rRNA and RLEP), and the human 18S rRNA gene as internal control. The limit of detection was estimated to be 2.29 copies of the M. leprae genome. Analytical specificity was evaluated using a panel of 20 other skin pathogenic microorganisms and Mycobacteria, showing no cross-reactivity. Intra- and inter-operator Cp variation was evaluated using dilution curves of M. leprae DNA or a synthetic gene, and no significant difference was observed between three operators in two different laboratories. The multiplex assay was evaluated using 97 patient samples with clinical and histopathological leprosy confirmation, displaying high diagnostic sensitivity (91%) and specificity (100%). Validation tests in an independent panel of 50 samples confirmed sensitivity and specificity of 97% and 98%, respectively. Importantly, assay performance remained stable for at least five months. Our results show that the newly developed multiplex qPCR effectively and specifically detects M. leprae DNA in skin samples, contributing to an efficient diagnosis that expedites the appropriate treatment.