RESUMO
The heat-shock protein 70 gene (hsp70) has been exploited for Leishmania species identification in the New and Old World, using PCR followed by restriction fragment length polymorphism (RFLP) analysis. Current PCR presents limitations in terms of sensitivity, which hampers its use for analyzing clinical and biological samples, and specificity, which makes it inappropriate to discriminate between Leishmania and other trypanosomatids. The aim of the study was to improve the sensitivity and specificity of a previously reported hsp70 PCR using alternative PCR primers and RFLPs. Following in silico analysis of available sequences, three new PCR primer sets and restriction digest schemes were tested on a globally representative panel of 114 Leishmania strains, various other infectious agents, and clinical samples. The largest new PCR fragment retained the discriminatory power from RFLP, while two smaller fragments discriminated less species. The detection limit of the new PCRs was between 0.05 and 0.5 parasite genomes, they amplified clinical samples more efficiently, and were Leishmania specific. We succeeded in significantly improving the specificity and sensitivity of the PCRs for hsp70 Leishmania species typing. The improved PCR-RFLP assays can impact diagnosis, treatment, and epidemiological studies of leishmaniasis in any setting worldwide.
Assuntos
Proteínas de Choque Térmico HSP70/genética , Leishmania/genética , Leishmania/isolamento & purificação , Leishmaniose/diagnóstico , Leishmaniose/parasitologia , Reação em Cadeia da Polimerase/métodos , Proteínas de Protozoários/genética , Primers do DNA/genética , Humanos , Leishmania/classificação , Parasitologia/métodos , Polimorfismo de Fragmento de Restrição , Sensibilidade e EspecificidadeRESUMO
INTRODUCTION: Species typing in leishmaniasis gains importance in diagnostics, epidemiology, and clinical studies. A restriction fragment length polymorphism (RFLP) assay of PCR amplicons from a partial heat-shock protein 70 gene (hsp70) had been described for the New World, allowing identification of some species. METHODS: Based on an initial in silico analysis of 51 hsp70 sequences, most of which we recently determined in the frame of a phylogenetic study, species-specific restriction sites were identified. These were tested by PCR-RFLP on 139 strains from 14 species, thereby documenting both inter- and intra-species variability. RESULTS: Our assay could identify Leishmania infantum, L. donovani, L. tropica, L. aethiopica, L. major, L. lainsoni, L. naiffi, L. braziliensis, L. peruviana, L. guyanensis, and L. panamensis by applying 2 subsequent digests. L. mexicana, L. amazonensis, and L. garnhami did not generate species-specific restriction fragment patterns. CONCLUSION: Currently no assay is available for global Leishmania species discrimination. We present a universal PCR-RFLP method allowing identification of most medically relevant Old and New World Leishmania species on the basis of a single PCR, obviating the need to perform separate PCRs. The technique is simple to perform and can be implemented in all settings where PCR is available.
Assuntos
Proteínas de Choque Térmico HSP70/genética , Leishmania/classificação , Leishmaniose/diagnóstico , Leishmaniose/parasitologia , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Fragmento de Restrição , Animais , Humanos , Leishmania/genética , Leishmania/isolamento & purificação , Leishmaniose Cutânea/diagnóstico , Leishmaniose Cutânea/parasitologia , Polimorfismo de Nucleotídeo Único , Especificidade da EspécieRESUMO
The in vitro antileishmanial effect of the essential oil from Chenopodium ambrosioides against Leishmania donovani was investigated. The product showed significant activity against promastigotes and amastigotes, with a 50% effective concentration of 4.45 and 5.1 microg/mL, respectively. The essential oil caused an irreversible inhibition of the growth of promastigotes after a treatment with 100 or 10 microg/mL for 1 or 24 h, respectively. The phagocytic activity of the macrophages was preserved at a concentration toxic to the parasite. The essential oil from C. ambrosioides may be a potential candidate drug to development a new agent to combat this parasitic disease.
Assuntos
Chenopodium ambrosioides/química , Leishmania donovani/efeitos dos fármacos , Óleos Voláteis/farmacologia , Animais , Leishmaniose Visceral/parasitologiaRESUMO
The existing difficulties in the treatment of leishmaniasis justify the testing of the effect of new products on parasite forms in the search of a therapeutic alternative for the parasitosis. Given the need of establishing a method to evaluate the activity of natural synthetic products in vitro under the Cuban conditions, this paper was aimed at defining the usefulness of p-nitrophenilphosphate as a chromogenic substance for quantification of parasites in plaques from 96 wells. To standardize this colorimetric method the stages of the parasite growth curve were set. The study of linearity and selection of the sample size, which was optional for these assays, showed that it was possible to obtain maximum linear determination coefficient with 20 mm. Likewise, the variation coefficient was compared with and without the chromogen and the effect of changes in culture medium on the reading of absorbances was analyzed. The set limit of quantification proved the need of using chromogen for the purposes of this paper and the general results allow to recommend this less subjective, simpler and quicker methodology to test products of interest in this field.
Assuntos
Compostos Cromogênicos , Água Doce/parasitologia , Leishmania/isolamento & purificação , Nitrofenóis , Compostos Organofosforados , Abastecimento de Água , Animais , Cuba , Meios de Cultura , Leishmania/crescimento & desenvolvimento , Modelos LinearesRESUMO
An immunoenzymatic system on solid phase for the detection of IgG antibodies in serum from chronic Chagasic patients was standardized. A protease from Trypanosoma cruzi (GP57/51KDa) was used as an antigen. The sensitivity, specificity and predictive values of the procedure were calculated taking into account the results obtained from conventional serology (CS), indirect immunofluorescence (IIF), indirect hemagglutination (IHA) and complement-mediated lysis test (CML) for the detection of anti-Trypanosoma cruzi antibodies in serum. Except one, the positive samples obtained by those techniques were also positive by our method. These results show that GP 57/51 is useful in the serodiagnosis of Chagas disease.
Assuntos
Antígenos de Protozoários , Doença de Chagas/diagnóstico , Cisteína Endopeptidases , Glicoproteínas , Trypanosoma cruzi/enzimologia , Animais , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Doença de Chagas/imunologia , Ensaios Enzimáticos Clínicos , Cisteína Endopeptidases/imunologia , Ensaio de Imunoadsorção Enzimática , Técnica Indireta de Fluorescência para Anticorpo , Glicoproteínas/imunologia , Humanos , Imunoglobulina G/sangue , Trypanosoma cruzi/imunologiaRESUMO
A serological study to detect antibodies to Toxocara canis in a group of 156 healthy children from City of Havana is reported for the first time in Cuba. An ELISA method was employed using excretion/secretion antigens obtained in our laboratory. Data on epidemiological factors surveyed in this group are presented. Positivity percentage was of 5.2%. Results are discussed.