Identification of Leishmania species by high-resolution DNA dissociation in cases of American cutaneous leishmaniasis.

BACKGROUND: American cutaneous leishmaniasis is an infectious dermatosis caused by protozoa of the genus Leishmania, which comprises a broad spectrum of clinical manifestations depending on the parasite species involved in the infections and the immunogenetic response of the host. The use of techniques for amplification of the parasites DNA based on polymerase chain reaction polymerase chain reaction and the recent application of combined techniques, such as high-resolution DNA dissociation, have been described as a viable alternative for the detection and identification of Leishmania spp. in biological samples. OBJECTIVES: To identify the Leishmania species using the polymerase chain reaction high-resolution DNA dissociation technique in skin biopsies of hospital-treated patients, and compare with results obtained by other molecular identification techniques. METHODS: A retrospective study assessing patients with suspected American cutaneous leishmaniasis seen at a hospital in São Paulo/Brazil was conducted. The paraffin blocks of 22 patients were analyzed by polymerase chain reaction high-resolution DNA dissociation to confirm the diagnosis and identify the species. RESULTS: Of the 22 patients with suspected American cutaneous leishmaniasis, the parasite was identified in 14, comprising five cases (35.6%) of infection by L. amazonensis, four (28.5%) by L. braziliensis, two (14.4%) by L. amazonensis+L. infantum chagasi, two (14.4%) by L. guyanensis, and one (7.1%) by Leishmania infantum chagasi. In one of the samples, in which the presence of amastigotes was confirmed on histopathological examination, the polymerase chain reaction high-resolution DNA dissociation technique failed to detect the DNA of the parasite. STUDY LIMITATIONS: The retrospective nature of the study and small number of patients. CONCLUSIONS: The method detected and identified Leishmania species in paraffin-embedded skin biopsies with a sensitivity of 96.4% and could be routinely used in the public health system.

Biochemical and biophysical properties of a highly active recombinant arginase from Leishmania (Leishmania) amazonensis and subcellular localization of native enzyme

Arginase (l-arginine amidinohydrolase, E.C. 3.5.3.1) is a metalloenzyme that catalyses the hydrolysisof l-arginine to l-ornithine and urea. In Leishmania spp., the biological role of the enzyme may beinvolved in modulating NO production upon macrophage infection. Previously, we cloned and characterizedthe arginase gene from Leishmania (Leishmania) amazonensis. In the presentwork,we successfullyexpressed the recombinant enzyme in E. coli and performed biochemical and biophysical characterizationof both the native and recombinant enzymes. We obtained KM and Vmax values of 23.9(±0.96)mM and192.3 mol/minmg protein (±14.3), respectively, for the native enzyme. For the recombinant counterpart,KM was 21.5(±0.90)mMand Vmax was 144.9(±8.9) mol/min mg. Antibody against the recombinantprotein confirmed a glycosomal cellular localization of the enzyme in promastigotes. Data fromlight scatteringand small angle X-ray scattering showed that a trimeric state is the active form of the protein.Wedetermined empirically that a manganesewash at room temperature is the best condition to purify activeenzyme. The interaction of the recombinant protein with the immobilized nickel also allowed us to confirmthe structural disposition of histidine at positions 3 and 324. The determined structural parametersprovide substantial data to facilitate the search for selective inhibitors of parasitic sources of arginase,which could subsequently point to a candidate for leishmaniasis therapy.