Evaluating the spatial distribution of Leishmania parasites in Colombia from clinical samples and human isolates (1999 to 2016).

In Colombia, nine species of parasites of the genus Leishmania circulate in more than 20 sand fly species, putting at risk of contracting the disease approximately 60% of the population. The Federico Lleras Acosta Dermatological Center, a reference center in Colombia, has been treating patients with cutaneous and mucosal leishmaniasis for more than 15 years, identifying the infecting Leishmania species from different clinical samples, and recording systematically all the epidemiological and geographic information related to each diagnosed patient. With this valuable information, the objective of this work was to perform a long term and large-scale study, aiming to identify the Leishmania species circulating in Colombia from clinical samples from 1999 to 2016, and to assess their current and potential spatial distribution. In all, four Leishmania species were identified in 688 samples from 183 municipalities distributed in 28 of the 32 departments of the country, and 387 records were georeferenced, from 20 departments. The most widespread species was L. (V.) braziliensis, showing new collection records, and the species related to areas with highest leishmaniasis transmission was L. (V.) panamensis. Ecological niche models were built for the three species that had more than 20 georeferenced records, showing a potential distribution for L. (V.) braziliensis on 42% of the national territory mainly in the interandean valleys, and the Orinoquia and Amazon regions. Leishmania (V.) guyanensis potential distribution covers 36% of Colombia continental territory with a spatial distribution similar to that of L. (V.) braziliensis. There was a marked tendency of L. (V.) panamensis to be distributed in the northwest of the country occupying 35% of the national area and mainly in areas of transformed ecosystems. Species were identified in patients from areas where the occurrence of cases was unprecedented, which suggests that the distribution of Leishmania may be greater than currently known. To improve the predictive capacity of the models, we suggest incorporating, in future studies, Leishmania samples from vectors and reservoirs that have a greater dependence on environmental variables. Our results are an important tool for health systems because they allow potential areas of transmission and information gaps to be identified.

Functional evaluation of gene silencing on macrophages derived from U937 cells using interference RNA (shRNA) in a model of macrophages infected with Leishmania (Viannia) braziliensis.

Leishmaniasis development is multifactorial; nonetheless, the establishment of the infection, which occurs by the survival and replication of the parasite inside its main host cell, the macrophage, is mandatory. Thus, the importance of studying the molecular mechanisms involved in the Leishmania-macrophage interaction is highlighted. The aim of this study was to characterize a cellular model of macrophages derived from U937 cells that would allow for the identification of infection phenotypes induced by genetic silencing with interference RNA in the context of macrophages infected with Leishmania (Viannia) braziliensis. The model was standardized by silencing an exogenous gene (gfp), an endogenous gene (lmna) and a differentially expressed gene between infected and non-infected macrophages (gro-ß). The silencing process was successful for the three genes studied, obtaining reductions of 88·9% in the GFP levels, 87·5% in LMNA levels and 74·4% for Gro-ß with respect to the corresponding control cell lines. The cell model revealed changes in the infection phenotype of the macrophages in terms of number of amastigotes per infected macrophage, number of amastigotes per sampled macrophage and percentage of infected macrophages as a result of gene silencing. Thus, this cell model constitutes a research platform for the study of parasite-host interactions and for the identification of potentially therapeutic targets.

Changes in Macrophage Gene Expression Associated with Leishmania (Viannia) braziliensis Infection.

Different Leishmania species cause distinct clinical manifestations of the infectious disease leishmaniasis. It is fundamentally important to understand the mechanisms governing the interaction between Leishmania and its host cell. Little is known about this interaction between Leishmania (Viannia) braziliensis and human macrophages. In this study, we aimed to identify differential gene expression between non-infected and L. (V) braziliensis-infected U937-derived macrophages. We deployed a whole human transcriptome microarray analysis using 72 hours post-infection samples and compared those samples with their non-infected counterparts. We found that 218 genes were differentially expressed between infected and non-infected macrophages. A total of 71.6% of these genes were down-regulated in the infected macrophages. Functional enrichment analyses identified the steroid and sterol/cholesterol biosynthetic processes between regulatory networks down-regulated in infected macrophages. RT-qPCR further confirmed this down-regulation in genes belonging to these pathways. These findings contrast with those from studies involving other Leishmania species at earlier infection stages, where gene up-regulation for this metabolic pathway has been reported. Sterol biosynthesis could be an important biological process associated with the expression profile of macrophages infected by L. (V.) braziliensis. Differential transcriptional results suggest a negative regulation of the genetic regulatory network involved in cholesterol biosynthesis.