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Pentavalent antimonials are the mainstay treatment against different clinical forms of leishmaniasis. The emergence of resistant isolates in endemic areas has led to treatment failure. Unraveling the underlying resistance mechanism would assist in improving the treatment strategies against resistant isolates. This study aimed to investigate the RNA expression level of glutathione synthetase (GS), Spermidine synthetase (SpS), trypanothione synthetase (TryS) genes involved in trypanothione synthesis, and thiol-dependent reductase (TDR) implicated in drug reduction, in antimony-sensitive and -resistant Leishmania tropica isolates. We investigated 11 antimony-resistant and 11 antimony-sensitive L. tropica clinical isolates from ACL patients. Drug sensitivity of amastigotes was determined in mouse macrophage cell line J774A.1. The RNA expression level in the promastigote forms was analyzed by quantitative real-time PCR. The results revealed a significant increase in the average expression of GS, SpS, and TrpS genes by 2.19, 1.56, and 2.33-fold in resistant isolates compared to sensitive ones. The average expression of TDR was 1.24-fold higher in resistant isolates, which was insignificant. The highest correlation coefficient between inhibitory concentration (IC 50 ) values and gene expression belonged to the TryS, GS, SpS, and TDR genes. Moreover, the intracellular thiol content was increased 2.17-fold in resistant isolates compared to sensitive ones and positively correlated with IC 50 values. Our findings suggest that overexpression of trypanothione biosynthesis genes and increased thiol content might play a key role in the antimony resistance of L. tropica clinical isolates. In addition, the diversity of gene expression in the trypanothione system and thiol content among L. tropica clinical isolates highlighted the phenotypic heterogeneity of antimony resistance among the parasite population., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Antimony is an important drug for the treatment of Leishmania parasite infections. In several countries, the emergence of drug-resistant Leishmania species has reduced the effectiveness of this drug. The mechanism of clinical drug resistance is unclear. The aim of this work was to identify mitochondrial proteome alterations associated with resistance against antimonial. A combination of cell fractionation, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and Label-Free Quantification was used to characterize the mitochondrial protein composition of Leishmania tropica field isolates resistant and sensitive to meglumine antimoniate. LC-MS/MS analysis resulted in the identification of about 1200 proteins of the Leishmania tropica mitochondrial proteome. Various criteria were used to allocate about 40% proteins to mitochondrial proteome. Comparative quantitative proteomic analysis of the sensitive and the resistant strains showed proteins with differential abundance in resistance species are involved in TCA and aerobic respiration enzymes, stress proteins, lipid metabolism enzymes, and translation. These results showed that the mechanism of antimony resistance in Leishmania spp. field isolate may be associated with alteration in enzymes involved in mitochondrial pathways.

Cutaneous leishmaniosis (CL) is mainly caused by Leishmania major (rural-type) and Leishmania tropica (urban-type). CL is a major health problem in many regions of the world, and it is associated with health complications and economic loss. The identification and differentiation of Leishmania species are critical because the prevention and control methods, as well as management and therapeutic strategies, are different for each type of CL. The present study aimed to identify the parasite species responsible for CL in the study area using ITS1 and HSP70- based PCR-RFLP methods. A total of 147 stained slides were prepared from samples collected from CL patients, and these slides were positive for amastigotes of Leishmania species on microscopic examination. Forty-three Giemsastained slides with 2+ to 4+ grades were selected for molecular studies for the identification of the Leishmania species. DNA was extracted from the selected slides for the molecular studies. The amplification of HSP70 and ITS1 genes was performed by the PCR method. The PCR products were digested with the HaeIII restriction enzyme, and banding patterns of all samples were compared with reference strains. Overall, patterns of all the samples were found to correspond to the reference strains of L. major based on RFLP-PCR targeting HSP70 and ITS1 genes of the parasite, demonstrating the dominance of L. major as the causative agent of zoonotic cutaneous leishmaniosis (zCL) in the study area. This area is endemic for zoonotic CL, and further studies are required to determine the reservoir and natural infection of sand flies in this county.

The mitochondrion of kinetoplastida has unique characteristics both in structure and function. To better understand the mitochondrial proteome of the Leishmania tropica promastigote stage, liquid chromatography coupled with mass spectrometry (LC/MS/MS) approach was used. In the wake of mitochondria isolation and purity validation, 1212 proteins were identified, among which approximately 44% of proteins belonged to the mitochondrial proteome. Several functions were enriched in mitochondrial proteome including tricarboxylic acid cycle and respiratory chain, protein folding, signalling, transport, lipid metabolism, amino acid, and nucleotide metabolism. Furthermore, the result of the present research was compared with the previous related studies. Gaining more information about vital metabolism of the cell and molecules can be used for therapeutic purposes., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Pentavalent antimonials remain the treatment of choice for all the clinical forms of leishmaniasis. The increasing rates of antimony resistance are becoming a serious health problem in treatment of anthroponotic cutaneous leishmaniasis (ACL). Accordingly, unraveling molecular markers is crucial for improving medication strategies and monitoring of drug-resistant parasites. Different studies have suggested the importance of genes involved in trypanothione metabolism and drug transport. In this regard, present study was designed to investigate the RNA expression level of five genes including γ-GCS, ODC, TRYR (involved in trypanothione metabolism), AQP1 (acts in drug uptake) and MRPA (involved in sequestration of drug) in sensitive and resistant Leishmania tropica isolates. Seven antimony-resistant and seven antimony-sensitive L. tropica clinical isolates were collected from ACL patients. Drug sensitivity test was performed on the samples as well as reference strains; afterwards, gene expression analysis was performed on clinical isolates by quantitative real-time PCR. The results revealed that the average expression level of AQP1 gene was decreased (0.47-fold) in resistant isolates compared to sensitive ones whereas MRPA (2.45), γ-GCS (2.1) and TRYR (1.97) was upregulated in resistant isolates. The average expression of ODC (1.24-fold) gene was not different significantly between sensitive and resistant isolates. Our findings suggest that AQP1, MRPA, GSH1 and TRYR can be considered as potential molecular markers for screening of antimony resistance in some L. tropica clinical isolates.

Leishmania RNA virus (LRV) was first detected in members of the subgenus Leishmania (Viannia), and later, the virulence and metastasis of the New World species were attributed to this virus. The data on the presence of LRV in Old World species are confined to Leishmania major and a few Leishmania aethiopica isolates. The aim of this study was to survey the presence of LRV in various Iranian Leishmania species originating from patients and animal reservoir hosts. Genomic nucleic acids were extracted from 50 cultured isolates belonging to the species Leishmania major, Leishmania tropica, and Leishmania infantum. A partial sequence of the viral RNA-dependent RNA polymerase (RdRp) gene was amplified, sequenced and compared with appropriate sequences from the GenBank database. We detected the virus in two parasite specimens: an isolate of L. infantum derived from a visceral leishmaniasis (VL) patient who was unresponsive to meglumine antimoniate treatment, and an L. major isolate originating from a great gerbil, Rhombomys opimus. The Iranian LRV sequences showed the highest similarities to an Old World L. major LRV2 and were genetically distant from LRV1 isolates detected in New World Leishmania parasites. We could not attribute treatment failure in VL patient to the presence of LRV due to the limited number of specimens analyzed. Further studies with inclusion of more clinical samples are required to elucidate the potential role of LRVs in pathogenesis or treatment failure of Old World leishmaniasis.

Background: Resistance to pentavalent antimonial drugs has become a serious problem in the treatment of cutaneous leishmaniasis in some endemic areas. Investigations on molecular markers involved in drug resistance are essential for monitoring of the disease. Leishmania-activated C kinase gene (LACK1) is involved in multiple central processes such as signal transduction. According to the probable role of the LACK1 gene in antimony resistance, we used real-time reverse transcription-polymerase chain reaction (PCR) to investigate the expression of this gene in clinical L. tropica strains, which were resistant or sensitive to meglumine antimoniate., Methods: We analyzed the expression level of LACK in 18 sensitive and 14 resistant L. tropica isolates collected from patients with anthroponotic cutaneous leishmaniasis. After cDNA synthesis, gene expression analysis was performed by quantitative real-time PCR using SYBR Green. In addition, the full length of the LACK gene from six reference strains was cloned and sequenced then deposited in the NCBI database to confirm our strains., Results: Real-time reverse transcription-PCR revealed that the average RNA expression level of LACK in isolates from unresponsive and responsive patients were 0.479 and 4.583, respectively, and expression of LACK was significantly downregulated (9.56-fold) in resistant isolates compared to sensitive ones., Conclusion: Results of the present study suggest the probable role of the LACK gene in antimony resistance. Moreover, it can be considered as a potential marker for monitoring antimony resistance in clinical isolates. However, further studies are required to exploit the biological functions of it in antimony resistance., (© 2016 The International Society of Dermatology.)

Unlabelled: Viscerotropic leishmaniasis (VTL) is a parasitic disease with non-specific manifestations caused by Leishmania tropica. Specific antigens produced by Viscerotropic leishmaniasis gene have been used for diagnosis of VTL. The aim of this study was to compare the expression level of VTL gene among the viscerotropic L. tropica isolates (n: 3) and visceral L. infantum isolates (n: 4). Also, the expression level was compared in L. tropica (n: 21) and L. major (n: 8) isolates, the main causes of cutaneous leishmaniasis in Iran by real time-RT-PCR. Results showed viscerotropic leishmaniasis gene was expressed in all 3 species; L. tropica, L. major and L. infantum. The most expression rate was in L. tropica and L. major as the cutaneous species and the lowest in visceral isolates including L. infantum and viscerotropic L. tropica strains respectively., Conclusion: Results revealed that VTL gene can play an important role in visceralization process of L. tropica although there are other mechanisms to keep parasite visceralized. According to these primary results, increased the expression level of VTL gene probably could contribute to inhibit the invasive behavior of Leishmania parasites. However, more experimental researches are needed to confirm this idea.

Pentavalent antimonial compounds have been the first line therapy for leishmaniasis; unfortunately the rate of treatment failure of anthroponotic cutaneous leishmaniasis (ACL) is increasing due to emerging of drug resistance. Elucidation of the molecular mechanisms operating in antimony resistance is critical for development of new strategies for treatment. Here, we used a cDNA-AFLP approach to identify gene(s) which are differentially expressed in resistant and sensitive Leishmania tropica field isolates. We identified five genes, aquaglyceroporin (AQP1) acts in drug uptake, ATP-binding cassette (ABC) transporter (MRPA) involved in sequestration of drug, phosphoglycerate kinase (PGK) implicated in glycolysis metabolism, mitogen activated protein kinase (MAPK) and protein tyrosine phosphatase (PTP) responsible for phosphorylation pathway. The results were confirmed using real time RT-PCR which revealed an upregulation of MRPA, PTP and PGK genes and downregulation of AQP1 and MAPK genes in resistant isolate. To our knowledge, this is the first report of identification of PTP and PGK genes potentially implicated in resistance to antimonials. Our findings support the idea that distinct biomolecules might be involved in antimony resistance in L. tropica field isolates., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The mainstay therapy against leishmaniasis is still pentavalent antimonial drugs; however, the rate of antimony resistance is increasing in endemic regions such as Iran. Understanding the molecular basis of resistance to antimonials could be helpful to improve treatment strategies. This study aimed to recognize genes involved in antimony resistance of Leishmania tropica field isolates. Sensitive and resistant L. tropica parasites were isolated from anthroponotic cutaneous leishmaniasis patients and drug susceptibility of parasites to meglumine antimoniate (Glucantime®) was confirmed using in vitro assay. Then, complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) and real-time reverse transcriptase-PCR (RT-PCR) approaches were utilized on mRNAs from resistant and sensitive L. tropica isolates. We identified 2 known genes, ubiquitin implicated in protein degradation and amino acid permease (AAP3) involved in arginine uptake. Also, we identified 1 gene encoding hypothetical protein. Real-time RT-PCR revealed a significant upregulation of ubiquitin (2.54-fold), and AAP3 (2.86-fold) (P<0.05) in a resistant isolate compared to a sensitive one. Our results suggest that overexpression of ubiquitin and AAP3 could potentially implicated in natural antimony resistance.

Background: Recent circumstantial evidence suggests that an increasing number of Iranian patients with cutaneous leishmaniasis are unresponsive to meglumine antimoniate (Glucantime), the first line of treatment in Iran. This study was designed to determine whether the clinical responses (healing, or non-healing) were correlated with the susceptibility of Leishmania parasites to Glucantime., Methods and Findings: In vitro susceptibility testing was first performed on 185 isolated parasites in the intracellular mouse peritoneal macrophage model. A strong correlation between the clinical outcome and the in vitro effective concentration 50% (EC50) values was observed. Parasites derived from patients with non-healing lesions had EC50 values at least 4-fold higher than parasites derived from lesions of healing patients. A selection of these strains was typed at the molecular level by pulsed-field gels and by sequencing the pteridine reductase 1 (PTR1) gene. These techniques indicated that 28 out of 31 selected strains were Leishmania tropica and that three were Leishmania major. The L. major isolates were part of a distinct pulsed-field group, and the L. tropica isolates could be classified in three related additional pulsed-field groups. For each pulsed-field karyotype, we selected sensitive and resistant parasites in which we transfected the firefly luciferase marker to assess further the in vitro susceptibility of field isolates in the monocyte cell line THP1. These determinations confirmed unequivocally that patients with non-healing lesions were infected with L. tropica parasites resistant to Glucantime. Additional characterization of the resistant isolates showed that resistance is stable and can be reversed by buthionine sulfoximine, an inhibitor of glutathione biosynthesis., Conclusions: To the authors' knowledge, this is the first report of proven resistant parasites contributing to treatment failure for cutaneous leishmaniasis and shows that primary Glucantime-resistant L. tropica field isolates are now frequent in Iran.

Background: Pentavalent antimonial has been a drug of choice against leishmaniasis, despite the emergence of treatment failure. Identification of resistance markers is urgently needed to design new therapeutic strategies. Iron-Superoxide dismutases (Fe-SODs) are antioxidant enzymes contributing to detoxify reactive oxygen species to prevent a cell from oxidative stress. Since antimonial compounds induce oxidative stress, in this survey, the expression of SOD genes was investigated to identify their expression pattern in clinical resistant isolates. Methods: This cross-sectional survey was done in Mashhad City, northeast of Iran during 2014 to 2019. The RNA expression level of mitochondrial (SODA) and glycosomal (SODB) superoxide dismutase was investigated in 25 antimony responsive (n=15) and unresponsive (n=10) anthroponotic cutaneous leishmaniasis (ACL) patients. Total RNA extraction and cDNA synthesis, the qRT-PCR approach was utilized to investigate the relative RNA expression level. Results: The transcript level of SODs was over-expressed in the most resistant isolates. Gene expression analysis demonstrated the over-expression of SODA and B by a factor of 3.8 and 4.81, respectively, in resistance isolates vs. sensitive ones. Conclusion: Aberrant expression of SODA/B in unresponsive parasites could potentially implicate in detoxifying antimony-induced oxidative stress. Moreover, SODs might be considered as potential predictive markers of the response to antimonials in ACL patients in endemic areas. [ABSTRACT FROM AUTHOR]

Despite the broad distribution of leishmaniasis among Iranians and animals across the country, little is known about the genetic characteristics of the causative agents. Applying both HSP70 PCR-RFLP and sequence analyses, this study aimed to evaluate the genetic diversity and phylogenetic relationships among Leishmania spp. isolated from Iranian endemic foci and available reference strains. A total of 36 Leishmania isolates from almost all districts across the country were genetically analyzed for the HSP70 gene using both PCR-RFLP and sequence analysis. The original HSP70 gene sequences were aligned along with homologous Leishmania sequences retrieved from NCBI, and subjected to the phylogenetic analysis. Basic parameters of genetic diversity were also estimated. The HSP70 PCR-RFLP presented 3 different electrophoretic patterns, with no further intraspecific variation, corresponding to 3 Leishmania species available in the country, L. tropica, L. major, and L. infantum. Phylogenetic analyses presented 5 major clades, corresponding to 5 species complexes. Iranian lineages, including L. major, L. tropica, and L. infantum, were distributed among 3 complexes L. major, L. tropica, and L. donovani. However, within the L. major and L. donovani species complexes, the HSP70 phylogeny was not able to distinguish clearly between the L. major and L. turanica isolates, and between the L. infantum, L. donovani, and L. chagasi isolates, respectively. Our results indicated that both HSP70 PCR-RFLP and sequence analyses are medically applicable tools for identification of Leishmania species in Iranian patients. However, the reduced genetic diversity of the target gene makes it inevitable that its phylogeny only resolves the major groups, namely, the species complexes. [ABSTRACT FROM AUTHOR]

Background: Cutaneous leishmaniasis (CL) is one of the most important neglected tropical diseases and a major public health challenge in Iran caused by Leishmania spp and transmitted by phlebotomine sand flies. The number of CL cases has shown an increasing pattern all over the country, including the district of Varamin, southeast of Tehran, Iran. This study aimed to identify the Leishmania spp isolated from CL patients using molecular methods in Varamin during 2012-2013. Methods: Exudate materials collected from the swollen edge of the skin lesions of 44 parasitological positive CL patients by disposable lancet. They were referred to Varamin Health Center by physician. The samples were subjected to molecular method for Leishmania species identification. Results: The digestion pattern of restriction enzyme revealed that 37 (84.1%) CL patients were infected with L. major and 7 (15.9%) were infected with L. tropica. They were mostly male than female. More than half of the patients (58%) had multiple lesions, and they were mostly observed on extremities, 34.1% on legs and 29.5% on hands. Le- sions were mostly of wet ulcerative type. Conclusion: Dominancy of L. major provides more evidence that Varamin District probably could be considered as Zoonotic Cutaneous Leishmaniasis (ZCL) areas. More investigation on other epidemiological aspects of disease is needed. [ABSTRACT FROM AUTHOR]

We describe a case of visceral leishmaniasis (VL) due to Leishmania tropica in a 50- year-old Iranian man lived in a VL-endemic area in southwest of Iran. The patient presented with a 3-month history of fever and splenomegaly. Clinical signs and serological findings were suggestive of VL. Spleen biopsy was taken from the patient and intracellular forms of Leishmania amastigotes was seen in Giemsa stained smears. The patient was treated with pentavalent antimonial compound with complete resolution of his systemic signs and symptoms. DNA was extracted from the microscopic slides of the spleen biopsy and the nagt (N-Acetylglucosamine-1- Phosphate Transferase) gene of Leishmania was PCR-amplified. Sequence analysis of the PCR product demonstrated that the case has 99% identity with those of available sequences of L. tropica. Intra-species variation within isolate was 0-0.1%; whereas, inter-species differences of the isolate with those of L. major and L. infantum was significantly higher. [ABSTRACT FROM AUTHOR]