Introduction: Leishmaniasis is one of the most common infectious diseases in tropical and subtropical areas in the world which is reporting from Iran and more than 90 countries all around the world. Generally primary treatment of leishmaniasis depends mainly on pentavalent antimonies including Glucantime and Pentostam and resistance to these compounds has been reported in recent years. Failure to treatment and decrease in effectiveness of Glucantime has been reported from Iran. Resistance to antimonial compounds may occur due to several mechanisms and several genes related to the resistance are detected, including efflux pomp genes MRPA and mdr1 that code ABC transporters which efflux drugs from the cell membrane or sequester metal-thiol conjugates.
The aim of this study is to detect mdr1 and MRPA genes in clinical isolates of Leishmania promastigotes.
Methods: In this study, during a period of 24 months, direct samples were taken from skin lesions of patients who referred to Center for Research and Training in Skin Diseases and Leprosy. Smears were prepared from the lesions and stained with Giemsa and directly examined for the presence of Leishmania amastigotes under the light microscope. Part of discharge was inoculated into biphasic culture media NNN (consisted of Agar, Rabbit's blood & liquid phase RPMI) or NNNN (consisted of Agar, Rabbit's blood, liquid phase Lockes) and after incubation were examined for the presence of promastigotes. Also for detection of Leishmania species, part of the discharge was transferred to vials containing sterile PBS for PCR test. DNA extraction was done by phenol- chloroform procedure from amastigotes of lesions or from promastigotes which were grown in liquid media RPMI enriched with 10 % FBS. The species of Leishmania (L. tropica or L. major) were detected by PCR using specific primer for ITS region. Then frequency of efllux genes MRPA and mdr1 was determined by using PCR method. For this purpose, specific primers targeted conserved regions were designed by using softwares. During the set up procedure, different concentrations of reaction mixture and temperatures were examined.
Results: In total 40 cutaneous leishmaniasis patients were included, from whom 40 strians of Leishmania were isolated. Twenty samples were used during set up procedure of PCRs and the remaining 20 isolates were used for molecular characterization of efflux pomp genes. From 20 isolates, 9 samples (45%) were positive in stained smear, 9 samples (45%) were positive in 3N/4N culture and 16 samples (80%) were positive in PCR. By PCR, 6 samples (30%) were characterized as L. major, 10 samples (50%) as L. tropica and 4 samples (20%) were unidentified. From 20 isolates, 19 samples (95%) were mdr1 positive, 10 samples (50%) were MRPA positive and 10 samples (50%) had both genes. Among MRPA positive samples, 4 were L. tropica and 5 were L. major, while among MDR positive samples, 10 were L. tropica and 5 were L. major
Conclusion: In this study, ABC transporter genes particularly mdr1, which previously was identified in experimentally induced resistance strains of Leishmania spp., were successfully identified in clinical isolates of Leishmania causing cutaneos leishmaniasis. Confirmation of the possible role of these genes in drug resistance requires further study.
Keywords: Cutaneous leishmaniasis; Drug resistance; Efflux pomps; Antimonial compounds; L. major; L. tropica