RESUMO
The present work evaluates sampling protocols, storage procedures, and DNA purification methods for Leishmania spp. detection and quantification in different biological samples. The efficiency of three preservation solutions, a phosphate buffer solution, an ethylenediaminetetraacetic acid (EDTA) buffer solution, and 70% ethanol, was compared in combination with three DNA extraction protocols: a commercial silica column kit, salting-out protein precipitation, and organic extraction with phenol-chloroform. Tissue samples from BALB/c mice experimentally infected with Leishmania (Leishmania) amazonensis, Leishmania (Viannia) braziliensis, or Leishmania (Leishmania) infantum were stored in the three preservation solutions and subsequently subjected to the three different DNA extraction methods. The extracted DNA was then used in real-time polymerase chain reaction (PCR) assays for the detection and quantification of parasite ribosomal small subunit DNA targets as well as mammalian glyceraldehyde-3-phosphate dehydrogenase (gapdh) targets. The results of the optimized protocols showed that the DNA extraction method did not influence test quality, but DNA from samples preserved with the EDTA buffer solution produced higher amounts of target amplicons. Based on these results, we concluded that samples from suspected cases of leishmaniasis for submission to molecular diagnostic procedures should be preferentially preserved in EDTA, followed by any one of the DNA purification methods evaluated.
Assuntos
Leishmania braziliensis , Leishmania infantum , Leishmaniose , Animais , Camundongos , DNA de Protozoário/genética , Ácido Edético , Leishmania braziliensis/genética , Leishmania infantum/genética , Mamíferos , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase em Tempo Real/métodosRESUMO
RESUMEN La leishmaniasis es una de las enfermedades tropicales más desatendidas, causada por el parásito Leishmania. En Paraguay, la especie responsable de la leishmaniasis cutánea es (LC) es L. (Viannia) braziliensis. Aquí se reporta un caso diagnosticado de Leishmaniasis, y análisis moleculares empleando reacción en cadena de la polimerasa - polimorfismos de restricción de fragmentos de restricción (PCR-RFLP) demostraron que el caso fue causado por L. (V.) lainsoni. Este es el primer registro de esta especie para Paraguay, con lo cual se extiende el rango de distribución conocido de la especie, unos 1.430 km al sur de localidades previamente conocidas. Se necesitan más estudios para conocer la incidencia real de esta especie en casos de LC en Paraguay, y para identificar reservorios naturales del parásito en la naturaleza.
ABSTRACT Leishmaniasis is one of the most neglected tropical diseases worldwide caused by the parasite Leishmania. In Paraguay the species responsible for cutaneous leishmaniasis (CL) is L. (Viannia) braziliensis. Here we report a case diagnosed with Leishmaniasis, and molecular analyses using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) demonstrate that the case was caused by L. (V.) lainsoni. This is the first record of this species for Paraguay, with which we extend the distribution range of the parasite 1,430 km southwards from the southernmost previous known locality. More studies are needed to know the actual incidence of this species in cases of CL in Paraguay, and to identify natural reservoirs in the wild.
RESUMO
American tegumentary leishmaniasis (ATL), an endemic anthropozoonosis in various countries in the world, is caused by parasites of the genus Leishmania. Despite reports on ATL reactivation as a result of immunosuppression, to the best of our knowledge, this paper describes the first case of ATL reactivation in its localized form (cutaneous leishmaniasis) associated with the administration of systemic corticosteroids. The possible action of corticosteroids on the host immune response to the parasite in patients with localized cutaneous leishmaniasis is discussed. This report demonstrates the possibility of ATL reactivation in patients using corticosteroids, an observation that should be considered in individuals treated with this medication.
