A flow cytometric technique was developed for detection of amastigotes of the rotozoan Leishmania infantm in human nonadherent monocyte-derived macrophages. The cells were fixed and permeabilized with paraformaldehyde-ethanol, and intracellular amastigotes were labeled with Leishmania lipophosphoglycan-specific monoclonal antibody. This method provides accuragte quantification of the infection rates in human macrophages compared to the rates obtained by the conventional microscopic technique, with the advantage that a large number of cells could be analyzed rapidly. The results demonstrated that labeling of intracellular amastigotes could reliably be used to evaluate the antileishmanial activities of conventional drugs such as meglumine antimoniate, amphotercin B, pentaminidine, and allopurinol.

They also established that various Leishmania species,mexicana and donovani, could also be detected by this technique in other host-cell models such as mouse peritoneal macrophages and suggested that the flow cytometric method could be a valid alternative to the conventional method. Leishmaniases are vector-borne parasitic diseases caused by protozoa of the genus Leishmania. These obligate intracellular parasites replicate in the parasitophorus vacuoles of macrophages and produce life-threatening disorders. The protozoa is widely distributed in tropical, subtropical, and Mediterranean countries. Several clinical syndromes are subsumed under the term leishmaniasis according to the localization of the parasites in mammalian tissues, notably: visceral, cutaneous, and mucosal leishmaniasis. Parasites of the species Leishmania infantum are identified as causative agents of the visceral type of leishmaniasis, and this is endemic in France. Since 1940, the generally accepted therapy for all forms of leishmanises consisted of pentavalent antimonial agents, used as first-line of clinical treatment. Later on various studies had been conducted through vitro host-cell models such as mouse peritoneal macrophages, human monocytes, or Chinese hamster overy cells, to study inection of mammalian cells with Leishmania parasites. In these models infection rates were usually measured by microscopic examination of adherent cells. In 1992, Ogunkolade et al developed an in vitro-model that permitted infection of human monocytes-derived macrophages by various species of Leishmania. This technique presented the advantage that adherent as well as free cells could be obtained easily and be infected in vitro by the promastigote stage of gthe parasite. On the other hand, frecent developments demonstratged that flow cytometry could be used to study infection of mammalian cells by various Leishmania species and established that amastigote-containing macrophages could be reliably be separated from non-infected cells by immunofluorecence. An investigation had been conducted for the possibility of using flow cytometry for the rapid and reliable detection of possible antileishmanial drugs. First, a flow cytometry assay was developed based on the staining of intracellular amastigotes with lipophophoglycan-specific monoclonal antibody in non-adherent human monocyte-derived macrophages, and then antileishmanial activities of antileishmanial drugs were studied. Drugs like meglumine antimoniate, pentamidine, allopurinol, and amphotericin B against parasites of the species of Leishmania infantum. Likewise the effect of meglumine against leishmania mexicana and donovani was studied in host cells-mouse peritoneal macrophages. Various assays were also done in human monocytes as well as mouse macrophages. Flow cytometry consists of an automated means of single-cell multiparametric analysis at a rate of several hundreds of cells per second with high degrees of accuracy and reproducibility. Since 1975, various techniques based on the use of fluorescent probes have been developed to provide quantitative information on the structural and functional parameters reflecting cell behavior.