RESUMO
Serological diagnosis of Trypanosoma cruzi infection is hampered by issues related to test specificity due to the cross-reactivity of most antigens with proteins of related parasites such as Leishmania spp. The recombinant calflagins are considered relevant antigens for the diagnosis of infection by Trypanosoma cruzi. In the present work, we describe two genes coding for putative calflagins in Leishmania major with the N-terminal moieties presenting high similarity with T. cruzi genes. This fact raised questions about their role in some cross-recognition of this antigen by sera from Leishmania spp.-infected individuals. The complete T. cruzi calflagin and two fragments of the protein, consisting of 146 amino acids of the N-terminal and 65 amino acids of the C-terminal regions, were expressed and evaluated against a panel of sera, which included well-characterized samples from T. cruzi, and Leishmania-infected patients. We were able to show that sera from Leishmania (Viannia) braziliensis-infected individuals recognized the recombinant full-length calflagin. Both the N-terminal and the complete protein presented the same high sensitivity (98.5% of sera from T. cruzi-infected patients was detected) but different specificities (94% and 98%, respectively, when evaluated against sera from people not infected by T. cruzi, including 15 sera from people infected with L. braziliensis). The C-terminal fragment presented low sensitivity (70%) but 100% specificity. We propose the use of these antigens in two sequential assays to optimize the serological diagnosis of T. cruzi infection in humans in geographic areas where Leishmania spp. infection is coendemic.
Assuntos
Proteínas de Ligação ao Cálcio/biossíntese , Doença de Chagas/diagnóstico , Proteínas de Protozoários/biossíntese , Trypanosoma cruzi/química , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/biossíntese , Antígenos de Protozoários/química , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/genética , Ensaio de Imunoadsorção Enzimática/métodos , Escherichia coli/metabolismo , Testes de Hemaglutinação/métodos , Humanos , Dados de Sequência Molecular , Engenharia de Proteínas , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas Recombinantes/biossíntese , Sensibilidade e Especificidade , Alinhamento de SequênciaRESUMO
In the present work we propose a simple method for affinity purification of the 67-kDa lectin-like glycoprotein (LLGP-67) from Trypanosoma cruzi, the causative agent of Chagas' disease. The LLGP-67, which presents galactose binding activity and participates in the host cell recognition process, was previously purified by methods based on its interaction with galactose residues on erythrocytic membranes. We describe herein results showing that this protein can be purified from T. cruzi in a direct way using non-derivatized agarose as a chromatographic ligand. We also demonstrate the relevance of LLGP-67 as an antigen for human diagnosis of chagasic infection. Sensitivity and specificity for this antigen were calculated, being 98 and 98.11% respectively.