Tape-disc-loop-mediated isothermal amplification (TD-LAMP) method as noninvasive approach for diagnosis of cutaneous leishmaniasis caused by L. tropica.

Cutaneous leishmaniasis (CL) is a parasitic disease caused by the bite of infectious female sand flies with high socioeconomic burdens. There is currently no non-invasive, point-of-care, diagnostic method with high sensitivity and specificity available for CL. We herein report the development of a non-invasive tape disc (TD) sampling method combined with a loop-mediated isothermal amplification (LAMP) assay using primer sets targeting kinetoplast DNA (kDNA) of Leishmania tropica (L. tropica) with a colorimetric readout for species-specific diagnosis of CL. We tested our Tape-Disc (TD)-LAMP method on a panel of skin samples collected by TD from 35 confirmed L. tropica patients, 35 healthy individuals and 35 patients with non-L. tropica infections. The detection limit of the TD-LAMP assay was determined as 1 fg (fg), and the assay sensitivity and specificity of 97 % and 100 % for L. tropica infection, respectively. This non-invasive, sensitive and rapid diagnostic method warrants further exploration of its use for differential diagnosis of CL in disease endemic settings.

Guidelines for the purification and characterization of extracellular vesicles of parasites.

Parasites are responsible for the most neglected tropical diseases, affecting over a billion people worldwide (WHO, 2015) and accounting for billions of cases a year and responsible for several millions of deaths. Research on extracellular vesicles (EVs) has increased in recent years and demonstrated that EVs shed by pathogenic parasites interact with host cells playing an important role in the parasite's survival, such as facilitation of infection, immunomodulation, parasite adaptation to the host environment and the transfer of drug resistance factors. Thus, EVs released by parasites mediate parasite-parasite and parasite-host intercellular communication. In addition, they are being explored as biomarkers of asymptomatic infections and disease prognosis after drug treatment. However, most current protocols used for the isolation, size determination, quantification and characterization of molecular cargo of EVs lack greater rigor, standardization, and adequate quality controls to certify the enrichment or purity of the ensuing bioproducts. We are now initiating major guidelines based on the evolution of collective knowledge in recent years. The main points covered in this position paper are methods for the isolation and molecular characterization of EVs obtained from parasite-infected cell cultures, experimental animals, and patients. The guideline also includes a discussion of suggested protocols and functional assays in host cells.

Isolation, characterization, and functional study of extracellular vesicles derived from Leishmania tarentolae.

Leishmania (L.) species are protozoan parasites with a complex life cycle consisting of a number of developmental forms that alternate between the sand fly vector and their host. The non-pathogenic species L. tarentolae is not able to induce an active infection in a human host. It has been observed that, in pathogenic species, extracellular vesicles (EVs) could exacerbate the infection. However, so far, there is no report on the identification, isolation, and characterization of L. tarentolae EVs. In this study, we have isolated and characterized EVs from L. tarentolae GFP+ (tEVs) along with L. major GFP+ as a reference and positive control. The EVs secreted by these two species demonstrated similar particle size distribution (approximately 200 nm) in scanning electron microscopy and nanoparticle tracking analysis. Moreover, the said EVs showed similar protein content, and GFP and GP63 proteins were detected in both using dot blot analysis. Furthermore, we could detect Leishmania-derived GP63 protein in THP-1 cells treated with tEVs. Interestingly, we observed a significant increase in the production of IFN-γ, TNF-α, and IL-1ß, while there were no significant differences in IL-6 levels in THP-1 cells treated with tEVs following an infection with L. major compared with another group of macrophages that were treated with L. major EVs prior to the infection. Another exciting observation of this study was a significant decrease in parasite load in tEV-treated Leishmania-infected macrophages. In addition, in comparison with another group of Leishmania-infected macrophages which was not exposed to any EVs, tEV managed to increase IFN-γ and decrease IL-6 and the parasite burden. In conclusion, we report for the first time that L. tarentolae can release EVs and provide evidence that tEVs are able to control the infection in human macrophages, making them a great potential platform for drug delivery, at least for parasitic infections.