RESUMO
All New World Leishmania species can cause cutaneous lesions, while only Leishmania (Viannia) braziliensis has been associated with mucosal metastases. Multilocus enzyme electrophoresis (MLEE) is the optimal standard for species identification but is slow and costly. New methods for species identification are needed to ensure proper identification and therapy. The coding regions of four metabolic enzyme markers in the MLEE typing method: mannose phosphate isomerase (MPI), malate dehydrogenase (MDH), glucose-6-phosphate isomerase (GPI), and 6-phosphogluconate dehydrogenase (6PGD), were analysed from seven species of New World Leishmania isolated from patients with either cutaneous or mucosal lesions to identify specific genetic polymorphisms responsible for the phenotypic variations observed in the MLEE typing scheme. We identified species-specific polymorphisms and determined that a combination of sequencing of the mpi and 6pgd genes was sufficient to differentiate among seven closely related species of New World Leishmania and among isolates of L. braziliensis shown previously to have atypical MLEE patterns. When DNA isolated from 10 cutaneous lesion biopsies were evaluated, the sequence typing method was 100% concordant with the published MLEE/monoclonal antibody identification methods. The identification of species-specific polymorphisms can be used to design a DNA-based test with greater discriminatory power that requires shorter identification times. When the causative agent of the disease is L. braziliensis, this method ensures correct species identification, even when the agent is a genetic variant. Proper identification could facilitate adequate treatment, preventing the onset of the disfiguring mucosal form of the disease.
Assuntos
Loci Gênicos , Leishmania/genética , Leishmania/isolamento & purificação , Leishmaniose Cutânea/diagnóstico , Animais , América Central , DNA de Protozoário/análise , DNA de Protozoário/genética , Variação Genética , Genoma de Protozoário , Glucose-6-Fosfato Isomerase/análise , Glucose-6-Fosfato Isomerase/genética , Humanos , Leishmania/enzimologia , Leishmaniose Cutânea/enzimologia , Leishmaniose Cutânea/parasitologia , Malato Desidrogenase/análise , Malato Desidrogenase/genética , Manose-6-Fosfato Isomerase/análise , Manose-6-Fosfato Isomerase/genética , Dados de Sequência Molecular , Fosfogluconato Desidrogenase/análise , Fosfogluconato Desidrogenase/genética , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA , América do Sul , Especificidade da EspécieRESUMO
Leishmanial organisms isolated from 24 patients with Andean cutaneous leishmaniasis (uta) and from 7 with sylvatic leishmaniasis in both cutaneous and mucosal forms were characterized on the basis of their isoenzyme profiles for 13 enzymes using both cellulose acetate (CA) and thin-layer starch gel (TLS) electrophoretic techniques. Malate dehydrogenase (MDH) after electrophoresis on CA or TLS and mannose phosphate isomerase (MPI) on TLS were the only enzymes of 13 examined which discriminated between the organisms from patients with uta (L. (V.) peruviana) and those with sylvatic leishmaniasis (L. (V.) braziliensis). Mannose phosphate isomerase gave more clear-cut and reproducible discrimination than did MDH on either TLS or CA, and it is suggested that MPI is a reliable enzyme marker that can be used in routine TLS electrophoresis to distinguish between L. (V.) peruviana and L. (V). braziliensis.