ABSTRACT
Leishmania infantum causes visceral leishmaniasis (VL) in the New World. The diagnosis of VL is confirmed by parasitological and serological tests, which are not always sensitive or specific. Our aim was to design new primers to perform a Polymerase Chain Reaction (PCR) for detecting L. infantum. Sequences of the minicircle kinetoplast DNA (kDNA) were obtained from GenBank, and the FLC2/RLC2 primers were designed. Samples of DNA from L. infantum, Leishmania amazonensis, Leishmania braziliensis, Leishmania guyanensis, Leishmania naiffi, Leishmania lainsoni, Leishmania panamensis, Leishmania major and Trypanosoma cruzi were used to standardize the PCR. PCR with FLC2/RLC2 primers amplified a fragment of 230 bp and the detection limit was 0.2 fg of L. infantum DNA. Of the parasite species assayed, only L. infantum DNA was amplified. After sequencing, the fragment was aligned to GenBank sequences, and showed (99%) homology with L. infantum. In the analysis of blood samples and lesion biopsy from a dog clinically suspected to have VL, the PCR detected DNA from L. infantum. In biopsy lesions from humans and dogs with cutaneous leishmaniasis, the PCR was negative. The PCR with FLC2/RLC2 primers showed high sensitivity and specificity, and constitutes a promising technique for the diagnosis of VL.
Subject(s)
DNA Primers/genetics , DNA, Kinetoplast/genetics , DNA, Protozoan/genetics , Dog Diseases/diagnosis , Leishmania infantum/isolation & purification , Leishmaniasis, Cutaneous/diagnosis , Leishmaniasis, Visceral/diagnosis , Animals , Dogs , Humans , Leishmaniasis, Cutaneous/veterinary , Leishmaniasis, Visceral/veterinary , Polymerase Chain Reaction , Sensitivity and SpecificityABSTRACT
SUMMARY Leishmania infantum causes visceral leishmaniasis (VL) in the New World. The diagnosis of VL is confirmed by parasitological and serological tests, which are not always sensitive or specific. Our aim was to design new primers to perform a Polymerase Chain Reaction (PCR) for detecting L. infantum. Sequences of the minicircle kinetoplast DNA (kDNA) were obtained from GenBank, and the FLC2/RLC2 primers were designed. Samples of DNA from L. infantum, Leishmania amazonensis, Leishmania braziliensis, Leishmania guyanensis, Leishmania naiffi, Leishmania lainsoni, Leishmania panamensis, Leishmania major and Trypanosoma cruzi were used to standardize the PCR. PCR with FLC2/RLC2 primers amplified a fragment of 230 bp and the detection limit was 0.2 fg of L. infantum DNA. Of the parasite species assayed, only L. infantum DNA was amplified. After sequencing, the fragment was aligned to GenBank sequences, and showed (99%) homology with L. infantum. In the analysis of blood samples and lesion biopsy from a dog clinically suspected to have VL, the PCR detected DNA from L. infantum. In biopsy lesions from humans and dogs with cutaneous leishmaniasis, the PCR was negative. The PCR with FLC2/RLC2 primers showed high sensitivity and specificity, and constitutes a promising technique for the diagnosis of VL.
RESUMO Leishmania infantum causa leishmaniose visceral (LV) no Novo Mundo. O diagnóstico de LV é confirmado por testes parasitológicos e sorológicos, os quais nem sempre são sensíveis ou específicos. Nosso objetivo foi desenhar novos iniciadores para realizar uma Reação em Cadeia da Polimerase (PCR) para detecção de L. infantum. Sequências do DNA do minicírculo do cinetoplasto (kDNA) foram obtidos do GenBank, e os iniciadores FLC2/RLC2 foram desenhados. Amostras de DNA de L. infantum, Leishmania amazonensis, Leishmania braziliensis, Leishmania guyanensis, Leishmania naiffi, Leishmania lainsoni, Leishmania panamensis, Leishmania major e Trypanosoma cruzi foram usados para padronizar a PCR. PCR com iniciadores FLC2/RLC2 amplificou um fragmento de 230 pb e detectou 0,2 fg de DNA de L. infantum.Das espécies de parasitos analisadas, somente DNA de L. infantum foi amplificado. Após sequenciamento, o fragmento foi analisado no GenBank, que mostrou homologia com L. infantum. Em análises de amostras de sangue e lesão de cão com suspeita clínica de LV, a PCR detectou DNA de L. infantum. Em amostras de lesão de humanos e cães com leishmaniose cutânea, a PCR foi negativa. A PCR padronizada com os iniciadores FLC2/RLC2 mostrou alta sensibilidade e especificidade, sendo técnica promissora para o diagnóstico de LV.
Subject(s)
Animals , Dogs , Humans , DNA Primers/genetics , DNA, Kinetoplast/genetics , DNA, Protozoan/genetics , Dog Diseases/diagnosis , Leishmania infantum/isolation & purification , Leishmaniasis, Cutaneous/diagnosis , Leishmaniasis, Visceral/diagnosis , Leishmaniasis, Cutaneous/veterinary , Leishmaniasis, Visceral/veterinary , Polymerase Chain Reaction , Sensitivity and SpecificityABSTRACT
Leishmanioses são doenças infecciosas com diferentes formas clínicas e prognóstico, portanto a identificação da espécie é importante. Nós avaliamos o desempenho dos iniciadores LBF1/LBR1específicos para L. (V.) braziliensis por PCR e comparamos com resultados de Leishmania spp identificadas por anticorpos monoclonais. Das 29 L. (V.) braziliensis identificadas por anticorposmonoclonais, 16 (53,3por cento) foram detectadas e 7 (63,6por cento) das 11 Leishmania spp não identificadas apresentaram a banda de 536 pb. Estes iniciadores identificaram 87,7por cento de Leishmania do serodema III. Estes iniciadores indicam uma pequena correlação entre os dois métodos usados e também sugerem a existência de uma variabilidade genética entre isolados de L. (V.) braziliensis da regiãonoroeste do estado do Paraná.
Subject(s)
Leishmania braziliensis/isolation & purification , Leishmaniasis , Polymerase Chain ReactionABSTRACT
American cutaneous leishmaniasis (ACL) has a wide geographical distribution, including all the states of Brazil. Parasitological and immunological tests are the most commonly used methods for the laboratorial diagnosis of ACL. The objective of this study was to find a specific and sensitive antigen to the diagnosis of ACL. To this end, promastigotes of Leishmania (Viannia) braziliensis were submitted to gel-filtration chromatography (Sephadex G100) and to ionic-change chromatography (DEAE-Sepharose Fast-Flow), and the antigen reactivity was evaluated by enzyme immunoassay (EIA-IgG). The results showed that the peak II of fraction 8 distinguished ACL patient sera from those of healthy individuals and individuals with other diseases (P<0.0001), presenting 85.41% sensitivity and 91.22% specificity. False-positive results were found for sera from Chagas disease patients (16.67%) and healthy individuals (10.42%). False-positive results were not detected for sera from patients with toxoplasmosis or paracoccidioidomycosis. The fraction obtained shows good sensitivity and specificity for ACL diagnosis and opens up new possibilities for the use of serology in laboratorial diagnosis, seroepidemiological studies and in the follow up of ACL treatment.
