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Leishmania, a protozoan parasite, is responsible for a group of diseases known as leishmaniases (Ross, 1903). They are transmitted by the bite of the phlebotomine sandly to humans and animals (Killick-Kendrick, 1999). The disease is spreading because of changes in several risk factors such as climatic changes, environmental changes and population movements (for economic or political reasons) which may lead to alterations in the range and densities of the vectors and reservoirs of the disease, increasing human exposure to infected sandflies (Dujardin, 2008).
Visceral Leishmaniasis (VL), which is the most severe form of the disease, is caused by Leishmania infantum and L. donovani and may lead to death in 95% of the cases if not treated. These two species differ in: the pathology of the disease they cause, vectors and reservoir hosts as well as in their geographical distribution. There is a worrying augmentation of Leishmania’s resistance to pentavalent antimonial drugs (1st line treatm ...
Leishmania, a protozoan parasite, is responsible for a group of diseases known as leishmaniases (Ross, 1903). They are transmitted by the bite of the phlebotomine sandly to humans and animals (Killick-Kendrick, 1999). The disease is spreading because of changes in several risk factors such as climatic changes, environmental changes and population movements (for economic or political reasons) which may lead to alterations in the range and densities of the vectors and reservoirs of the disease, increasing human exposure to infected sandflies (Dujardin, 2008).
Visceral Leishmaniasis (VL), which is the most severe form of the disease, is caused by Leishmania infantum and L. donovani and may lead to death in 95% of the cases if not treated. These two species differ in: the pathology of the disease they cause, vectors and reservoir hosts as well as in their geographical distribution. There is a worrying augmentation of Leishmania’s resistance to pentavalent antimonial drugs (1st line treatment). However, alternative drugs have been developed for the successful treatment of the disease (Murray, 2004). First line treatment includes the use of liposomal Amphotericin B which may cause manifestations due to its acute toxicity (Ashford, 2000). Miltefosine (hexadecyl phosphocholine), the first oral drug against leishmaniasis, presents promising success (www.virbac.gr; Sundar, 2002).
Multi-drug resistance (MDR), is a condition enabling a disease – causing organism, or a cell, to resist substances of a wide variety of drugs (Singh, 2006). Today, MDR constitutes a major problem in the treatment of leishmaniasis and other infectious diseases and cancers.
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Cancer cell lines can acquire cross-resistance to a remarkably wide range of compounds which have no obvious structural or functional similarities. The cross-resistance seems to be due to the decreased accumulation of the drug, or the fluorescent dye (e.g Rhodamine – 123, Rhod - 123) in the cell, and has been attributed to alterations in the cell plasma membrane. An over-expressed plasma membrane glycoprotein of relative molecular mass (Mr) 170,000 Da (the P - glycoprotein Pgp - 170) is consistently found in different multidrug-resistant human and animal cell lines, in tumours and in various parasites. Consequently, it has been postulated that Pgp directly or indirectly mediates multidrug resistance (Riordan, 1985).
Prior the development of new drugs, it is important to investigate potential mechanisms underlying drug resistance which may include increased efflux or decreased influx of the drug.
The aim of this work was to assess and compare the biological and structural characteristics and the drug resistance of 10 Leishmania isolates, in vitro.
For this, 2 L. donovani and 8 L. infantum isolates, were used. The 10 strains were isolated from patients (2 L. donovani) and from dogs (8 L. infantum), from Cyprus, in the Laboratory of Clinical Bacteriology, Parasitology, Zoonoses and Geographical Medicine, Faculty of Medicine, University of Crete.
These isolates were characterized as “resistant: 5” (1 L. donovani and 4 L. infantum) and “sensitive: 5” (1 L. donovani and 4 L. infantum), according to the efflux rate of Rhod – 123 of the promastigote stage of each isolate, during preliminary studies using flow cytometry.
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Studies were carried out on the 10 isolates regarding their: a) biological characteristics such as the growth kinetics of their culture in vitro, the in vitro infectivity of the promastigotes (the infectious stage of the parasite for the vertebrate host) and the time required by the promastigotes to transform into axenic amastigotes in vitro (the form representing the intracellular form of the parasite in the vertebrate host) as well as the time required by the axenic amastigotes to transform into the promastigotes stage, b) the resistance expressed by the parasite to the 3 drugs used for the treatment of leishmaniasis (Glucantime, Amphotericin B ηαζ Miltefosine) and c) structural characteristics: comparison of the proteinic profile of the 10 isolates and the detection of the efflux pump Pgp – 170, by Western Blotting (WB).
The biological characteristics studied are probably related to the infectivity of the isolates to man, and the aim of the study was to characterize each isolate according to their infectivity and their resistance to drugs and to investigate how these factors are correlated with the structural characteristics studied (proteomic profile and the presence of the efflux pump), the disease pathology caused to the host and the geographical distribution of the isolate.
The two complementary methods used to study population growth (flow cytometry and the haemocytometer), showed consistent results for the 10 Leishmania isolates studied. The 2 L. donovani (H1, H2) isolates had lower growth rate, similar infectivity and required longer period for their promastigotes to transform into axenic amastigotes and reversely, in correlation to the 8 L. infantum (D1 – D8) isolates that had higher growth, presented diversity on their infectivity rates and required shorted period for their promastigotes to transform into axenic amastigotes, and reversely.
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All 10 isolates were resistant to Glucantime, a drug extensively used as a standard treatment against leishmaniasis in human and dogs in many countries. However, studying the resistance of a “sensitive” (H1) and 4 “resistant” (H2, D3, D4, D5) isolates on the main Glucantime compounds (SbV and SbIII), the results were consistent to those arouse during the preliminary studies.
The 5 “resistant” (H2, D3, D4, D5, D8) and the 2 out of 5 “sensitive” isolates (D1, D6) demonstrated some resistance to Liposomal Amphotericin B, a drug which substituted Glucantime as 1st line drug in many countries, whilst all 10 isolates showed no resistance to Miltefosine, a new drug used for the treatment of leishmaniasis.
For the proteomic analysis, 2 “sensitive” (H1, D2) and 2 “resistant” (H2, D3) isolates were studied. The isolate H2 (L. donovani) which was characterized as “resistant” during the preliminary studies was the only strain in which Heat Shock 70 kDa protein (HSP 70) was detected. The D3 (L. infantum) is a “resistant” isolate in which 2 HSP 70 - related mitochondrial precursor and 1 putative uncharacterized protein were detected. Two unidentified proteins were detected in all 4 isolated but over-expressed in “resistant” isolates. The Pgp – 170 efflux pump was detected in all 5 “resistant” isolates characterized as “resistant” during preliminary studies, whilst its expression was qualitatively higher in L. infantum isolates.
In conclusion, a) The use of CFSE vital stain and flow cytometry for the determination of the proliferation capacity of isolates is rapid, precise, sensitive and non-costly, b) The results of growth kinetics show that promastigotes of different species and strains have different division potential
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with L. donovani, isolated from patients, lower than L. infantum, isolated from dogs, c) the infectivity rate was different among the 10 isolates, d) the results of the, in vitro, transformation of promastigotes into axenic amastigotes and reversely, show that the species need different periods with L. donovani, longer than L. infantum, e) Glucantime cannot any longer be effectively used due to the multidrug resistance phenomenon (MDR) Leishmania parasites have induced, f) 70% of the isolates studied in vitro, developed some resistance in Amphotericin B, g) Miltefosine is a very effective drug against leishmaniasis, but its veterinary use should be avoided in areas of zoonotic leishmaniasis, where the dog is the reservoir for the parasite, since this might accelerate the emergence of drug resistance, h) the detection of Pgp – 170 and HSP 70 only in “resistant” isolates (Pgp – 170: H2, D3, D4, D5, D8 and HSP 70: H2, D3), supports that flow cytometry can safely be used to study the parasite‟s drug resistance, i) flow cytometry detects Pgp using a relatively small number of parasites (<100,000) whilst WB needs greater amount and longer time and procedure.
These data highlight the large effort that has been invested in understanding the changes associated with drug-resistance in Leishmania. Nevertheless, it is hoped that knowledge of such variety of physiological mechanisms (such as the efflux rate of Rhod – 123, the MDR, Pgp – 170, HSP 70 and promastigote - amastigote differentiation), will be translated into a more effective treatment and control of leishmaniasis.
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