Μελέτη της μολυσματικότητας και της αντοχής στελεχών Leishmania infantum, από την Αττική, την Κρήτη και την Κύπρο, στα αντιλεϊσμανιακά φάρμακα και η γεωγραφική τους κατανομή βάσει των χαρακτηριστικών τους

Abstract

Meglumine antimoniate is a first line drug for the treatment of visceral leish-maniasis. During the last years, isolates of Leishmania, the parasite responsi-ble for the disease, have developed resistance to this as well as other anti-leishmanial drugs because Leishmania possesses the MDR gene responsible for multidrug resistance. The aim of the project was the study of the pheno-menon of multidrug resistance in Leishmania with qualitative and quantitative tests using isolates from dogs and patients from Attica, Crete and Cyprus. The virulence of the isolates was also studied using human monocytes from a patient with acute monocytic leukemia (THP-1 cell line). The resistance to drugs of 199 Leishmania isolates from Attica, Crete and Cyprus was studied by flow cytometry. Five resistant and five sensitive isolates were selected based on the rate of efflux of the fluorescent probe Rhodamine 123 from the parasite body. Human macrophages were infected with the 10 Leishmania isolates and the transmembrane efflux pump, the glycoprotein Pgp 170 KDa, was marked using the monoclonal antibody C219 after double immunofluorescence. Microscopic analysis by confocal micro-scopy revealed the P-glycoprotein 170 KDa on the membrane of the amasti-gote body and the intensity of the fluorescence signal was related to the re-sistance of each strain as it was revealed by flow cytometry. Transmission electron microscopy, using one resistant and one sensitive strain, after immu-nogold labeling of the Pgp 170 KDa, showed that the number of pumps re-lates to the resistance of the strain. This was further demonstrated when the strains were made resistant, after incubation in an antileishmanial drug, which induced the production of more pumps by the parasite. The results of the study showed that the greater the rate of efflux of Rhoda-mine 123 in an isolate, during flow cytometry, the greater the fluorescence of the Pgp signal in the confocal microscope and the greater the number of Pgp efflux pumps in the transmission electron microscope (resistant strains). Thus, the results confirmed the important role of the Pgp efflux pump in the pheno-menon of multidrug resistance and the importance of flow cytometry as a tool for the study of the resistance of Leishmania isolates for the prediction of the success of the treatment in humans and dogs. The results of drug resistance for each isolate as well as its infectivity poten-tial were mapped for each study area and related to geographic and other fac-tors.