The effects of Leishmania RNA virus 2 (LRV2) on the virulence factors of L. major and pro-inflammatory biomarkers: an in vitro study on human monocyte cell line (THP-1).

BACKGROUND: Cutaneous Leishmaniasis (CL) is a parasitic disease with diverse outcomes. Clinical diversity is influenced by various factors such as Leishmania species and host genetic background. The role of Leishmania RNA virus (LRV), as an endosymbiont, is suggested to not only affect the pathogenesis of Leishmania, but also impact host immune responses. This study aimed to investigate the influence of LRV2 on the expression of a number of virulence factors (VFs) of Leishmania and pro-inflammatory biomarkers. MATERIALS AND METHODS: Sample were obtained from CL patients from Golestan province. Leishmania species were identified by PCR (LIN 4, 17), and the presence of LRV2 was checked using the semi-nested PCR (RdRp gene). Human monocyte cell line (THP-1) was treated with three isolates of L. major with LRV2 and one isolate of L. major without LRV2. The treatments with four isolates were administered for the time points: zero, 12, 24, 36, and 48 h after co-infection. The expression levels of Leishmania VFs genes including GP63, HSP83, and MPI, as well as pro-inflammatory biomarkers genes including NLRP3, IL18, and IL1ß, were measured using quantitative real-time PCR. RESULTS: The expression of GP63, HSP83, and MPI revealed up-regulation in LRV2 + isolates compared to LRV2- isolates. The expression of the pro-inflammatory biomarkers including NLRP3, IL1ß, and IL18 genes in LRV2- were higher than LRV2 + isolates. CONCLUSION: This finding suggests that LRV2 + may have a probable effect on the Leishmania VFs and pro-inflammatory biomarkers in the human macrophage model.

Effect of Leishmania RNA virus 2 on virulence factors and cytokines gene expression in a human macrophage infected with Leishmania major: A preliminary study.

Cutaneous leishmaniasis (CL) is one of the most important infectious parasitic diseases in the world caused by the Leishmania parasite. In recent decades, the presence of a virus from the Totiviridae family has been proven in some Leishmania species. Although the existence of LRV2 in the Old world Leishmania species has been confirmed, almost no studies have been done to determine the potential impact of LRV2 on the immunopathogenicity of the Leishmania parasite. In this preliminary study, we measured the expression of target genes, including Glycoprotein 63 (gp63), Heat Shock Protein 70 (hsp70), Cysteine Protease b (cpb), Interleukin 1 beta (IL-1ß), IL8 and IL-12 in LRV2 positive Leishmania major strain (LRV2+L. major) and LRV2 negative L. major strain (LRV2-L. major). We exposed THP-1, a human leukemia monocytic cell line, to promastigotes of both strains. After the initial infection, RNA was extracted at different time points, and the relative gene expression was determined using a real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Findings showed that the presence of LRV2 in L. major was able to increase the expression of gp63, hsp70, and cpb genes; also, we observed lower levels of expression in cytokine genes of IL-1ß, IL-8, IL-12 in the presence of LRV2+, which are critical factors in the host's immune response against leishmaniasis. These changes could suggest that the presence of LRV2 in L. major parasite may change the outcome of the disease and increase the probability of Leishmania survival; nevertheless, further studies are needed to confirm our results.

Leishmania RNA virus 2 (LRV2) exacerbates dermal lesions caused by Leishmania major and comparatively unresponsive to meglumine antimoniate treatment.

PURPOSE: The present study investigated the possible role of Leishmania RNA virus 2 (LRV2) in the severity of dermal lesions and treatment failure due to Leishmania major. METHODS: The drug susceptibility of 14 clinical isolates of L.major, including resistant (n = 7) and sensitive (n = 7) isolates, was checked in the J774A.1 macrophage cell line. The presence of LRV2 among isolates was investigated by the RdRp gene and semi-nested PCR. Moreover, 1 × 106 sensitive L. major LRV2+ and LRV2- promastigotes were inoculated subcutaneously into the base tails of the 40 BALB/c mice divided into 4 groups (n = 10 in each group), including clinical LRV2+, clinical LRV2-, positive control LRV2+ and negative control LRV2-. The groups were infected with a unique isolate. The lesion size and parasite burden were evaluated. RESULTS: Sensitive and resistant isolates were determined by the drug susceptibility method. A higher presence of LRV2 was observed among MA-resistant isolates (6/7) compared with susceptible isolates (4/7), which was not statistically significant (P = 0.237). On the other hand, a comparison of the lesion sizes between the LRV2+ and LRV2- BALB/c mice groups revealed that the mean size of the lesion in the LRV2+ groups was significantly higher than the LRV2- (P = 0.034). In the same direction, there was an increased parasite burden in mice inoculated with LRV2+ groups compared with the LRV2- BALB/c mice groups (P = 0.002). CONCLUSIONS: Our findings showed that the presence of LRV2 could be one of the factors contributing to exacerbating CL. Although we found a higher presence of LRV2 in the resistant isolates, it seems that further investigations are recommended to determine the detailed association between lesions' aggravation and being comparatively unresponsive to treatment.

Identification of immunodominant proteins of Leishmania infantum by immunoproteomics to evaluate a recombinant multi-epitope designed antigen for serodiagnosis of human visceral leishmaniasis.

Visceral leishmaniasis (VL) is a protozoan disease caused by Leishmania infantum in the Mediterranean region including Iran. In 95% of cases, the disease can be fatal if not rapidly diagnosed and left untreated. We aimed to identify immunoreactive proteins of L. infantum (Iranian strain), and to design and evaluate a recombinant multi-epitope antigen for serodiagnosis of human VL. To detect the immunoreactive proteins of L. infantum promastigotes, 2DE immunoblotting technique was performed using different pooled sera of VL patients. The candidate immunoreactive proteins were identified using MALDI-TOF/TOF mass spectrophotometry. Among 125 immunoreactive spots detected in 2-DE gels, glucose-regulated protein 78 (GRP78), ubiquitin-conjugating enzyme E2, calreticulin, mitochondrial heat shock 70-related protein 1 (mtHSP70), heat shock protein 70-related protein, i/6 autoantigen-like protein, ATPase beta subunit, and proteasome alpha subunit 5 were identified. The potent epitopes from candidate immunodominant proteins including GRP78, mtHSP70 and ubiquitin-conjugating enzyme E2 were then selected to design a recombinant antigenic protein (GRP-UBI-HSP). The recombinant antigen was evaluated by ELISA and compared to direct agglutination test for detection of anti L. infantum human antibodies. We screened 34 sera of VL patients from endemic areas and 107 sera of individuals without L. infantum infection from non-endemic area of VL. The recombinant protein-based ELISA provided a sensitivity of 70.6% and a specificity of 84.1%. These results showed that GRP78, ubiquitin-conjugating enzyme E2, and mtHSP70 proteins are potential immunodominant targets of the host immune system in response to the parasite and they can be considered as potential candidate markers for diagnosis purposes.

Comparative mitochondrial proteomics of Leishmania tropica clinical isolates resistant and sensitive to meglumine antimoniate.

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.

Mitochondrial proteome profiling of Leishmania tropica.

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.

Identification of Leishmania species using N-acetylglucosamine-1-phosphate transferase gene in a zoonotic cutaneous leishmaniasis focus of Iran.

BACKGROUND & OBJECTIVES: Ilam province is one of the oldest known endemic foci of zoonotic cutaneous leishmani- asis (CL) in Iran; and the recent studies have shown an increasing trend in the number of cases from the region. This study was aimed to investigate the parasite species and genetic diversity of isolates obtained from CL patients based on the N-acetylglucosamine-1-phosphate transferase (nagt) gene. METHODS: Exudate materials were collected from the swollen margin of the skin lesions of the patients suspected with CL who were referred to health centers laboratory of Mehran, Dehloran, Ilam and Malekshahi cities in the Ilam province. Demographic data were collected through a questionnaire. Smears were stained and examined microscopically. In total, 62 parasitologically positive samples were subjected to PCR-RFLP of nagt gene for identification of Leishmania species, in addition to genetic diversity investigation. RESULTS: Nearly, half of the positive cases were referred from Mehran followed by Dehloran City (40.4%). These included people from different age groups (1 to 73 yr), with majority being male (66.1%). The common site of lesions was hand (48.4%). Half of the patients had multiple lesions; most of them were wet ulcerative type. A 1450-60 bp band of the nagt gene was amplified from all the samples. Digestion patterns of acetyl-coenzyme A carboxylase 1 (ACC1) enzyme were similar to what expected for Leishmania major. No difference was observed at the nucleotide acid level or resulting amino acid in nine sequenced samples on the basis of phylogenetic analyses. However, intra- species differences (0.0015) were observed amongst the L. major isolates of present study and the other parts of Iran. INTERPRETATION & CONCLUSION: The findings of this study demonstrated that the main causative agent of CL in Ilam Province is L. major, and there is no considerable heterogeneity among the L. major isolates. Moreover, nagt gene proved to be an efficient marker for differentiating Leishmania species. Further studies with more samples need to be carried out to achieve a more comprehensive result on the genetic variation of L. major isolates.

High resolution melting analysis as an accurate method for identifying Leishmania infantum in canine serum samples.

BACKGROUND & OBJECTIVES: Leishmania (L.) infantum is the principal agent of visceral leishmaniasis (VL) in the Mediterranean and American regions. So far different molecular methods including high resolution melting (HRM) analysis have been developed for detecting and identifying L. infantum infection. HRM assay is an automted molecular method which detects and identifies different genus and species of infectious agents. This study aimed to diagnose and identify Leishmania infection caused by L. infantum species using real-time PCR coupled with HRM assay in the serum samples in comparison with anti-L. infantum antibodies obtained using direct agglutination test (DAT), in domestic and wild canines of northeastern Iran. METHODS: Serum samples of 15 foxes, 14 jackals, seven domestic dogs and three wolves were collected in some villages around Shirvan and Bojnourd districts from the northeast regions of Iran during 2014-15. Initially, all the collected serum samples were tested by DAT for the detection of anti-L. infantum antibodies. Afterwards, genomic DNA was extracted from the samples and tested by real-time PCR-HRM analysis targeting hsp70, ITS1 and gp63 genes. The level of agreement between DAT and HRM assay were analysed statistically. RESULTS: Out of the 39 serum samples, eight showed anti-L. infantum antibodies at titre 1: 80 while only one of them showed anti-L. infantum antibodies at titre 1 : 160. All the nine seropositive samples showed positive results with HRM analysis. Additionally, three DAT negative serum samples were also found positive in the HRM technique. Altogether, 12 out of the 39 DNA samples showed positive results in HRM analysis. Among the three gene sequences used, gp63 was best for separation and identification of species. INTERPRETATION & CONCLUSION: HRM analysis targeting hsp70, ITS1 and gp63 genes can be used as a highly sensitive technique for the screening and early detection of L. infantum infection in the wild and domestic canines. It has higher accuracy than DAT and allows detection and discrimination of different Leishmania species responsible for the Leishmaniases.

Genetic Diversity and Phylogenetic Analysis of the Iranian Leishmania Parasites Based on HSP70 Gene PCR-RFLP and Sequence Analysis.

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.

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.

Inter- and Intraspecific Variations of Leishmania Strains Isolated from Patients with Cutaneous and Visceral Leishmaniases in Fars Province, South of Iran.

BACKGROUND: Cutaneous and visceral leishmaniases are present in Fars Province in the south of Iran. The current study aimed to evaluate the inter- and intragenic diversities of Leishmania species isolated from patients with leishmaniasis in Fars Province, using PCR-based analyses and DNA sequencing of the N-acetylglucosamine-1-phosphate transferase (nagt) gene. METHODS: Clinical samples were taken from the skin lesions of 120 individuals with clinical suspicion of cutaneous leishmaniasis (CL) referred to the major health centers of Shiraz. Along with microscopic examination, a part of each sample was used for in vitro cultivation. DNA was extracted from the cultured parasites and the nagt gene was PCR-amplified. For RFLP analysis, the PCR product of the nagt gene was digested with the Acc1 restriction enzyme. Moreover, the PCR products of 23 isolates were sequenced and analyzed, using MEGA5. RESULTS: From the 120 patients with clinical suspicion of CL, 110 (91.7%) cases were found to be positive by direct microscopy while 77 (64.1%) of the cultures were positive. Digestion of the PCR product with the Acc1 restriction enzyme detected L. major in 57 out of the 77 (74.1%) and L. tropica, in 20 out of the 77 (25.9%) cases with CL. Phylogenetic analysis grouped the Leishmania isolates into 3 main clades, representing L. major, L. infantum, and L. tropica, encompassing 2, 2, and 2 haplotypes, respectively. Within the clades, the L. tropica intraspecies divergence was more pronounced in L. major. CONCLUSION: The findings of this study demonstrated that the causative agent of CL in Fars Province was mainly L. majorz and that there was considerable heterogeneity between the Leishmania species and also within the L. major isolates.

Visceral Leishmaniasis in Rural Areas of Alborz Province of Iran and Implication to Health Policy.

Visceral leishmaniasis (VL) or kala-azar mainly affects children in endemic areas. This study was conducted to determine the seroprevalence of VL using direct agglutination test (DAT) in children living in rural districts of Alborz Province located 30 km from Tehran capital city of Iran. Multi-stage cluster random sampling was applied. Blood samples were randomly collected from 1,007 children under 10 years of age in the clusters. A total of 37 (3.7%) of the studied population showed anti-Leishmania infantum antibodies with titers of ≥1:800. There was a significant association between positive sera and various parts of the rural areas of Alborz Province (P<0.002). Two children with anti-Leishmania infantum antibodies titers of ≥1:3,200 indicated kala-azar clinical features and treated with anti-leishmaniasis drugs in pediatric hospital. The findings of this study indicated that Leishmania infection is prevalent in rural areas of Alborz Province. Therefore, it is necessary to increase the awareness and alertness among physicians and public health managers, particularly in high-risk rural areas of the province in Iran.

Recognition of Immunoreactive Proteins in Leishmania infantum Amastigote-Like and Promastigote Using Sera of Visceral Leishmaniasis Patients: a Preliminary Study.

PURPOSE: Visceral leishmaniasis (VL) is a systemic and parasitic disease that is usually fatal if left untreated. VL is endemic in different parts of Iran and is caused mainly by Leishmania infantum. This study aimed to recognition immunoreactive proteins in amastigote-like and promastigote stages of L. infantum (Iranian strain) by antibodies present in the sera of VL patients. METHODS: Total protein extract from amastigote-like and promastigote cells was separated by two-dimensional electrophoresis (2DE). To detect the immunoreactive proteins, 2DE immunoblotting method was performed using different pools of VL patients' sera. RESULTS: Approximately 390 and 430 protein spots could be separated in 2DE profiles of L. infantum amastigote-like and promastigote stages, respectively. In immunoblotting method, approximately 295 and 135 immunoreactive proteins of amastigotes-like reacted with high antibody titer serum pool and low antibody titer serum pool, respectively. Approximately 120 and 85 immunoreactive proteins of promastigote extract were recognized using the high antibody titer sera pool and low antibody titer sera, respectively. CONCLUSION: The present study has recognized a number of antigenic diversity proteins based on the molecular weight and pH in amastigote-like and promastigote stages of L. infantum. These results provide us a new concept for further analysis development in the field of diagnosis biomarkers and vaccine targets.

A Geomedical Survey: Is There an Association Between Climatic Conditions and Leishmania Species Distribution in Iran During the Years 1999-2021?

PURPOSE: Iran is among the high-risk leishmaniasis regions in the world. WHO recommends the use of GIS as an ideal tool for healthcare authorities to predict the evolution of a disease, delimit the risk of outbreaks and identify critical areas. The aim of this research is to find the association between the main species of Leishmania (L. major, L. tropica, L. infantum) dispersion and climatic variables in Iran. METHODS: All molecular-based reports of leishmaniasis from Iran between 1999 and 2021 were gathered from reliable medical sources. Meteorological data (air and soil temperatures, annual rainfall and humidity) of the country along the study period were obtained from the Iranian Climatological Research Centre. The data concerning species distribution and climatic conditions during this period were moved to a base-map through raster layers using ArcGIS 10.4.1 software. The relationship between parasitological and climatic models was examined using ANOVA. RESULTS: High risk area maps, based on the cut-off thresholds, were generated for Leishmania major, L. tropica and L. infantum. According to the molecular-based reports, the L. major distribution was significantly related to all climatic variables, while L. tropica was merely related to rainfall and humidity, and the L. infantum distribution was significantly associated with rainfall, soil and air temperatures. CONCLUSION: The association between climatic conditions and Leishmania species distribution in Iran has been confirmed. Consequently, both, the relationship between climatic conditions and the geographical distribution of Leishmania species, and the use of GIS to better understand the spatial epidemiology of leishmaniasis, have been reaffirmed.

Insights into the trypanothione system in antimony-resistant and sensitive Leishmania tropica clinical isolates.

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 (IC50) 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 IC50 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.

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.

Proteomics investigation of human sera for determination of postoperative indicators of pulmonary cystic echinococcosis.

BACKGROUND: Cystic echinococcosis (CE)/hydatidosis is an important zoonotic parasitic disease caused by the larval stage of Echinococcus granulosus. The disease is a major health problem all over the world. Finding specific and sensitive biomarkers for follow-up of CE in patients after surgery is essential. Using proteomics methods, the present study aimed to evaluate post-surgical treatment by finding probable biomarker/s in the serum of human lungs CE. METHODS: A total of 24 human sera were tested. These sera included eight confirmed lung/s CE patients sera before surgery (BS), eight sera 12 months post-surgery (12MPS) as well as eight control sera from healthy people. Proteomics methods including 2DE and LC-MS/MS were performed on the specimens followed by bioinformatics analysis. Differentially expressed proteins (DEP) were detected and, separately integrated with protein-protein interaction (PPI) data to construct the PPI network. RESULTS: A total of 171 protein spots were detected in three groups including BS, 12MPS, and control groups; of which a total of 106 DEP have been expressed based on fold changes > = 2 and p-value < 0.05. More analysis was performed and a total of 10 protein spots were selected for identification by mass spectrometry showing the following proteins: APOA1, BGN, SPP2, EAF1, ACOXL, MRPL55, MCTP2, SEPTIN1, B4GALNT1, and ZNF843. Based on centrality parameters of the PPI network (degree and betweenness) five Hub-bottlenecks proteins with significant centrality values were found including APOA1, BGN, SPP2, EAF1, and ACOXL. CONCLUSION: This study showed five proteins as hub-bottleneck proteins; of which APOA1 was more prominent. It can be concluded that a change in expression of this protein in patients' sera could be used as an indicator tool for the achievement of lungs CE surgical therapy.

Amphotericin B-Loaded Extracellular Vesicles Derived from Leishmania major Enhancing Cutaneous Leishmaniasis Treatment through In Vitro and In Vivo Studies.

Background: Recent studies have shown an increasing number of patients with cutaneous leishmaniasis (CL) who do not respond to pentavalent antimonials as the first line of treatment for CL. Nanocarriers such as extracellular vesicles (EVs) are efficient vehicles that might be used as drug delivery systems for the treatment of diseases. Therefore, we aimed to isolate and characterize the EVs of Leishmania major, load them with Amphotericin B (AmB), and investigate the toxicity and efficacy of the prepared drug form. Methods: The EVs of L. major were isolated, characterized, and loaded with amphotericin B (AmB), and the EVs-Amphotericin B (EVs-AmB) form was synthesized. Relevant in vitro and in vivo methods were performed to evaluate the toxicity and efficacy of EVs-AmB compared to the control. Results: The anti-leishmanial activity of the EVs-AmB showed a higher percentage inhibition (PI%) (P = 0.023) compared to the AmB at different concentrations and time points. Obtained data showed a significant increase in the lesion size and parasite load in the lesion, PBS, and EVs mice groups in comparison with EVs-AmB, AmB, and Glucantime groups (P < 0.05), EVs-AmB had a significant decrease in lesion sizes in comparison with AmB (P < 0.05). Results showed that EVs-AmB decreased its toxicity to the kidneys and liver (P < 0.05). Conclusion: EVs-AmB improved the efficacy of AmB in mouse skin lesions and reduced hepatorenal toxicity. Furthermore, EVs could be a promising nanoplatform for the delivery of AmB in CL caused by L. major.

Assessment of genetic markers for multilocus sequence typing (MLST) of Fasciola isolates from Iran.

BACKGROUND: Several markers have been described to characterise the population structure and genetic diversity of Fasciola species (Fasciola hepatica (F. hepatica) and Fasciola gigantica (F. gigantica). However, sequence analysis of a single genomic locus cannot provide sufficient resolution for the genetic diversity of the Fasciola parasite whose genomes are ∼1.3 GB in size. OBJECTIVES: To gain a better understanding of the gene diversity of Fasciola isolates from western Iran and to identify the most informative markers as candidates for epidemiological studies, five housekeeping genes were evaluated using a multilocus sequence typing (MLST) approach. METHODS: MLST analysis was developed based on five genes (ND1, Pepck, Pold, Cyt b and HSP70) after genomic DNA extraction, amplification and sequencing. Nucleotide diversity and phylogeny analysis were conducted on both concatenated MLST loci and each individual locus. A median joining haplotype network was created to examine the haplotypes relationship among Fasciola isolates. RESULTS: Thirty-three Fasciola isolates (19 F. hepatica and 14 F. gigantica) were included in the study. A total of 2971 bp was analysed for each isolate and 31 sequence types (STs) were identified among the 33 isolates (19 for F. hepatica and 14 for F. gigantica isolates). The STs produced 44 and 42 polymorphic sites and 17 and 14 haplotypes for F. hepatica and F. gigantica, respectively. Haplotype diversity was 0.982 ± 0.026 and 1.000 ± 0.027 and nucleotide diversity was 0.00200 and 0.00353 ± 0.00088 for F. hepatica and F. gigantica, respectively. There was a high degree of genetic diversity with a Simpson's index of diversity of 0.98 and 1 for F. hepatica and F. gigantica, respectively. While HSP70 and Pold haplotypes from Fasciola species were separated by one to three mutational steps, the haplotype networks of ND1 and Cyt b were more complex and numerous mutational steps were found, likely due to recombination. CONCLUSIONS: Although HSP70 and Pold genes from F. gigantica were invariant over the entire region of sequence coverage, MLST was useful for investigating the phylogenetic relationship of Fasciola species. The present study also provided insight into markers more suitable for phylogenetic studies and the genetic structure of Fasciola parasites.