Developing molecular tools for the study of emerging parasites in Visceral Leishmaniasis

Abstract

Leishmaniasis is a neglected disease caused by more than 20 species of protozoa Leishmania, which are transmitted to humans through sandfly vectors. Visceral Leishmaniasis (VL) in Brazil is caused by Leishmania infantum, a serious disease that affects organs such as the spleen, bone marrow, liver and lymph nodes and can be fatal when not properly diagnosed and/or treated. Brazil is one of the endemic countries for Leishmaniasis and has a high prevalence rate for VL in the Northeast region. The treatment is done with amphotericin and pentavalent antimonials, but studies demonstrate the resistance of these parasites to these drugs and susceptibility may be associated with the immunological condition of the host and possible co-infections by other pathogens. Recently, a non-Leishmania parasites isolated in culture (promastigote) of an atypical and fatal case of HVL in HIV-negative patients were identified. Through phylogenomic analyses of clinical isolates of the same patient (obtained from bone marrow aspirate and skin lesions), we found that these protozoa are more similar to Crithidia fasciculata, a monoxenic species of trypanosomatid that parasites exclusively culicidae insects. Molecular phylogenetic analyses using nucleotide sequences of ribosomal RNA 18S region (SSU-rRNA) and more recently genomic data from other clinical isolates of VL from the same region showed the same result for several different cases, i.e., Crithidia-like parasites being isolated from patients diagnosed with VL. Therefore, there is an urgent need to develop methods of correct identification of the species involved in the infection. The detection method need to be species-specific, in order to investigate possible cases of co-infection. It is extremely important for patients and for public health to perform accurate diagnosis and appropriate clinical management. Furthermore, it is necessary to experimentally evaluate the potential for infection of Crithidia-like parasites and against the background of co-infection with L. infantum. Therefore, it is necessary to develop molecular tools directed to these species to understand and elucidate these severe and atypical cases of recurrent HVL in an endemic area of Northeastern Brazil. Thus, in this project will standardize a method of detection and quantification of Crithidia-like parasite and L. infantum by real-time qPCR, in addition to construct a Crithidia-like strain expressing fluorescent reporter gene to evaluate infections by this parasite. These tools will be important to understand the outcome of these infections. In addition to the qPCR method can be applied as a new alternative for molecular diagnosis. (AU)