Detection and molecular identification of leishmania RNA virus (LRV) in Iranian Leishmania species.

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.

Identification of antimony resistance markers in Leishmania tropica field isolates through a cDNA-AFLP approach.

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.

Overexpression of ubiquitin and amino acid permease genes in association with antimony resistance in Leishmania tropica field isolates.

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.

Treatment Failure in Cutaneous Leishmaniasis Patients Referred to the School of Public Health, Tehran University of Medical Sciences during 2008-2017.

BACKGROUND: Cutaneous leishmaniasis (CL) is a vector borne disease predominantly found in tropical and subtropical countries, including Iran. For more than 6 decades, pentavalent antimonials have been used successfully worldwide for the treatment of leishmaniasis, but over the past few years, clinical resistance to these medications has increased. In this study, we evaluated CL patients who did not show any desirable responses to the anti-leishmanial treatment within a 10-year period (2008 to 2017). METHODS: All patients from different parts of Iran suspected of having cutaneous leishmaniasis, who were referred to the laboratory of leishmaniosis in Tehran University of Medical Sciences from 2008-2017 were parasitological examined. RESULTS: During this period, a total of 1480 suspected CL patients were referred to the laboratory of leishmaniosis. Samples from 655 patients (70.8%) suspected of having CL were positive microscopically. The failure rate in patients treated with anti-leishmaniasis medications for a minimum of three complete treatment periods was 1.83% (12 cases). There was no association between the number and size of skin lesions and patient characteristics. Also, the route of drug administration had no significant effect on the number and size of lesions. CONCLUSION: In the present study, treatment failure was found in some confirmed CL patients treated with meglumine antimoniate. Over the past few years, it seems that had been increased in resistance to these medications. So, a review of the correct implementation of the treatment protocol and/or a combination therapy may be helpful in preventing an increase in the rate of treatment failure.

Comparison of p27 Gene Expression of Promastigote and Amastigote Forms of Leishmania major (MRHO/IR/75/ER) by Real-time RT-PCR.

BACKGROUND: Cutaneous leishmaniasis (CL) is one of the world health problems. Leishmania major is the etiological agent of zoonotic cutaneous leishmaniasis (ZCL). Promastigote and amastigote are two morphological forms of Leishmania parasites that express different proteins and p27 is an important gene encoding cytochrome c oxidase (COX) component. P27 gene expresses a 27 kDa protein that essential in ATP synthesis. This study aimed to compare p27 gene expression in promastigote and amastigote forms in Iranian strain of L. major (MRHO/IR/75/ER). METHODS: This study was conducted in 2015. Clinical isolates of CL patients from north, center, west and south parts of Iran were collected and identified by PCRRFLP. After RNA extraction of promastigotes and amastigotes and cDNA synthesis, the expression level of p27 gene was compared by real-time RT-PCR. RESULTS: By comparison of expression level between amastigote and promastigote forms of Iranian strain of L. major, up-regulation of p27 gene (2.73 fold) was observed in amastigotes. Moreover, there was no significant difference in p27 gene expression between L. major isolates. CONCLUSION: p27 gene and protein can be considered as a target in recombinant vaccine production and treatment process.

Expression, purification and in vitro refolding of the recombinant truncated Saposin-like protein 2 antigen for development of diagnosis of human fascioliasis.

Early diagnosis of fascioliasis is critical in prevention of injury to the liver and bile ducts. Saposin-like protein (FhSAP-2) is probably the most ideal antigen of Fasciola hepatica for development of ELISA kits. SAP-2 has a conserved tertiary structure containing three disulfide bonds and conformational epitopes. Therefore, antigenicity of SAP-2 is greatly depends on disulfide bond formation and proper folding. We produced the recombinant truncated SAP-2 (rtSAP-2) in the SHuffle® T7 and Rosetta strain of Escherichia coli, in soluble and insoluble forms, respectively and purified by immobilized metal affinity chromatography (IMAC). The refolding process of denatured rtSAP-2 was performed using dialysis and dilution methods in the presence of chemical additives, along with reduced/oxidized glutathione (in vitro). Physicochemical studies, including non-reducing gel electrophoresis, Ellman's assay, Western blotting and ELISA showed the most antigenicity and likely correct folding of rtSAP-2, which was obtained by dialysis method. An IgG ELISA test was developed using rtSAP-2 refolded by dialysis and compared with excretory/secretory products of parasite with 52 positive fascioliasis samples, 79 other parasitic samples and 70 negative controls samples. The results exhibited 100% sensitivity and 98% specificity for rtSAP-2, also, 100% and 95.3% for excretory/secretory (E/S) antigen, respectively. In conclusion, it is suggested that rtSAP-2 with the correct folding could be used as a candidate antigen for detection of human fascioliasis.

Expression analysis of viscerotropic leishmaniasis gene in Leishmania species by real-time RT-PCR.

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.

Expression analysis of activated protein kinase C gene (LACK1) in antimony sensitive and resistant Leishmania tropica clinical isolates using real-time RT-PCR.

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.

Diagnosing Malaria Cases Referred to the Malaria Reference Laboratory in Tehran University of Medical Science, Iran.

BACKGROUND: The number of malaria cases is declining worldwide; however, it remains as a serious health problem. Diagnosing unusual cases is the most important issue to manage the problem. This study designed to describe the number of falciparum and vivax malaria infected patients referred to Malaria Reference Laboratory in Tehran University of Medical Science from 2000 to 2012. METHODS: A retrospective study was conducted based on the collected questionnaires from each patient referred to the laboratory. Diagnosing results and demographic information for positive cases were analyzed using SPSS software. Problematic cases were evaluated for any difficulties in diagnosis or in clinical signs. Scanning and molecular methods were performed whenever there was an atypical case referred to the laboratory. Some of the samples had various difficulties for diagnosing such as presence of fussed gametocytes and schizonts of Plasmodium falciparum in peripheral blood and CCHF like hemoragic disorders. RESULTS: Plasmodium vivax caused a large proportion of the cases (76.1%) in contrast with P. falciparum that included smaller proportion (22.8%) and the rest (1.1) belonged to mixed infection. Most of the positive cases (69.6%) were belonged to Afghani people. Men (94.6%) showed more infection than women (5.4%), moreover the most infection (44.5%) was seen at a range of 21-30 yr. CONCLUSION: In the case of existing atypical issues to diagnose, it is needed to perform more precise microscopical examination beyond the current standard conditions. Sometimes molecular method is required to verify the exact agent of the disease.

Comparative Proteomic Profiling of Leishmania tropica: Investigation of a Case Infected with Simultaneous Cutaneous and Viscerotropic Leishmaniasis by 2-Dimentional Electrophoresis and Mass Spectrometry.

BACKGROUND: Viscerotropic leishmaniasis caused by Leishmania tropica poses a significant problem in the diagnosis and treatment management. Since differential gene expression is more important in outcome of the infection, we employed proteomic approach to identify potential proteins involved in visceralization of L. tropica. METHODS: The proteomes profiling of L. tropica isolated from cutaneous and visceral tissues of one host were compared by 2-DE/MS proteomics study. Moreover, the transcript level of some identified proteins was confirmed using real-time RT-PCR. RESULTS: Of the 700 protein spots that were detected reproducibly on each gel, 135 were found to be differentially expressed (P≤ 0.05). Most of responsive proteins in visceral isolate changed in less abundant compared to cutaneous isolate. Among differentially expressed proteins, 56 proteins were confidently identified and classified according to the biological process. The largest groups consist of proteins involved in carbohydrate metabolism and protein synthesis. Most of the identified proteins, which implicated in energy metabolism, cell signaling and virulence were down-regulated, whereas some proteins that have a role in protein folding, antioxidant defense and proteolysis were up-regulated in visceral form. Moreover, the transcript level of some identified proteins such as co-chaperon was confirmed using real-time RT-PCR. CONCLUSION: L. tropica probably uses different mechanisms for survival and multiplication in viscera to establish viscerotropic leishmaniasis. The current study provides some clues into the mechanisms underlying the dissemination of L. tropica .

Comparison of the Proteome Profiling of Iranian isolates of Leishmania tropica, L. major and L. infantum by Two-Dimensional Electrophoresis (2-DE) and Mass-spectrometry.

BACKGROUND: The mechanisms of virulence and species differences of Leishmania parasites are under the influence of gene expression regulations at posttranscriptional stages. In Iran, L. major and L. tropica are known as principal agents of cutaneous leishmaniasis, while L. infantum causes visceral leishmaniasis. METHODS: As a preliminary study, we compared the proteome mapping of the above three Iranian isolates of Leishmania species through the 2-dimension electrophoresis (2-DE), and identified the prominent proteins by Liquid Chromatography (LC) mass spectrometry. RESULTS: We reproducibly detected about 700 protein spots in each species by using the Melanie software. Totally, 264 proteins exhibited significant changes among 3 species. Forty nine protein spots identified in both L. tropica and L. major were similar in position in the gel, whereas only 35 of L. major proteins and 10 of L. tropica proteins were matched with those of L. infantum. Having identified 24 proteins in the three species, we sought to provide possible explanations for their differential expression patterns and discuss their relevance to cell biology. CONCLUSION: The comparison of proteome profiling pattern of the 3 species identified limit up and limit down regulated or absent /present proteins. In addition, the LC-MS data analysis showed that most of the protein spots with differential abundance in the 3 species are involved in cell motility and cytoskeleton, cell signaling and vesicular trafficking, intracellular survival / proteolysis, oxidative stress defense, protein synthesis, protein ubiquitination / proteolysis, and stress related proteins. Differentially proteins distributed among the species maybe implicated in host pathogenecity interactions and parasite tropism to cutaneous or visceral tissue macrophages.

Identifying differentially expressed genes in trophozoites and cysts of Acanthamoeba T4 genotype: Implications for developing new treatments for Acanthamoeba keratitis.

Acanthamoeba T4 genotype is the most prevalent genotype associated with amoebic keratitis. Acanthamoeba keratitis therapy is difficult due to transformation of trophozoite to cyst stage, which hinders the treatment of the disease. Although encystation assists the organism to survive against the chemotherapeutic compounds, the precise mechanism of encystation remains poorly understood. The purpose of this work was to identify differentially expressed genes in Acanthamoeba T4 genotype which might be useful for understanding of the encystment process and may thus help develop more efficient treatment. The mRNA profile of trophozoite and cyst of Acanthamoeba T4 genotype isolated from a soft contact lens wearer were analyzed using a cDNA amplified fragment length polymorphism (cDNA-AFLP) technique. Subsequently, a real time reverse transcriptase-PCR was performed to validate the cDNA-AFLP results. Three genes, heat shock protein70 (hsp70), actin-I and elongation factor-1alpha (EF-1α) were differentially expressed during Acanthamoeba differentiation. An in silico result predicted that transformation of trophozoite to cyst could be mediated through their cooperation with the protein partners interaction. Taken together, our experimental and bioinformatics findings suggested potential functions of hsp70, EF-1α and actin-I in differentiation of Acanthamoeba T4 genotype which may be useful in the design of an efficient therapeutic strategy in AK.

Identification and phylogenetic relationship of Iranian strains of various Leishmania species isolated from cutaneous and visceral cases of leishmaniasis based on N-acetylglucosamine-1-phosphate transferase gene.

The identity of Iranian Leishmania species has been resolved to some extent by some genetic markers. In this study, based on N-acetylglucosamine-1-phosphate transferase (nagt) gene, we further elucidated the identity and phylogeny of the prevalent species in this country. DNAs of 121 isolates belonging to cutaneous leishmaniasis (CL) patients, canine visceral leishmaniasis (CVL) cases, and Rhombomys opimus rodents were amplified by targeting a partial sequence of nagt gene. All the amplicons were analyzed with restriction fragment length polymorphism (RFLP) using Acc1 enzyme, and 49 amplicons representing different reservoir hosts were sequenced and aligned with similar sequences from GenBank database. The RFLP analysis revealed that 41 CL patients were infected Leishmania tropica and 36 with Leishmania major. Among 10 CVL isolates, 6 were identified as Leishmania infantum and 4 as L. tropica. Amongst 34 rodents' isolates, 11 and 23 isolates exhibited patterns similar to those of L. major, and L. tropica/Leishmania turanica, respectively. The sequencing results from all CL patients, CVL cases, and 4 reservoir rodents were in agreement with RFLP analysis and showed 99-100% homologies with the registered species of L. major, L. tropica, and L. infantum from Turkey, Tunisia, Iraq and Israel. Of the 7 rodent isolates exhibiting RFLP patterns similar to L. tropica/L. turanica, 3 exhibited the highest homologies (99-100%) with L. turanica and 4 with Leishmania gerbilli. The 49 nagt DNA sequences were grouped into five clusters representing L. major, L. tropica, L. infantum, L. turanica and L. gerbilli species, encompassing 19 haplotypes. No correlation was observed between intraspecies divergence and geographic distribution of haplotypes. The L. tropica haplotypes exhibited more homologies with those of L. infantum than L. major (97.2% vs. 96.9%), a probable indication to the potential ability of L. tropica to visceralize. Characterization of Iranian Leishmania isolates using nagt gene allowed unambiguous identification of five prevalent species with a high-resolution phylogeny.

Downregulation of Calcineurin Gene Is Associated with Glucantime(®) Resiatance in Leishmania infantum.

BACKGROUND: Pentavalent antimonials are the first line drugs for the treatment of leishmaniasis. Unresponsiveness of Leishmania spp. to antimonial drugs is a serious problem in some endemic areas. Investigations on molecular mechanisms involved in drug resistance are essential for monitoring and managing of the disease. Cal-cineurin is an essential protein phosphatase for number of signal transduction pathways in eukaryotic cells and it has a mediated role in apoptosis. This study aimed to determine of biomarker(s) in Glucantime(®) resiatance strain of L. infan-tum. METHODS: We used cDNA amplified fragment length polymorphism (cDNA-AFLP) and real time-RT PCR assays to compare gene expression profiles at the mRNA levels in resistant and susceptible L. infantum field isolates. RESULTS: The cDNA-AFLP results showed downlegulation of calcineurin in resis-tant isolate in comparison with susceptible one. Significant downregulation of cal-cineurin (0.42 fold) (P<0.05) was found in resistant isolate compared to susceptible one by Real time-RT PCR. CONCLUSION: This is the first report of calcineurin implication in Glucantime(®) drug resistance of field (natural) isolate of L. infantum. Downregulation of calcineurin could protect parasites from antimonial-induced apoptosis.