RESUMEN
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.
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Leishmania , Leishmaniasis Cutánea , Leishmaniavirus , Virus ARN , Humanos , Proteína con Dominio Pirina 3 de la Familia NLR , Monocitos , Interleucina-18 , Leishmaniavirus/genética , Virus ARN/genética , BiomarcadoresRESUMEN
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.
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Leishmania major , Leishmaniasis Cutánea , Virus ARN , Humanos , Citocinas/genética , Expresión Génica , Interleucina-12/genética , Leishmania major/genética , Leishmaniasis Cutánea/genética , Leishmaniasis Cutánea/microbiología , Macrófagos/microbiología , Virus ARN/patogenicidad , Factores de Virulencia/genéticaRESUMEN
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.
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Antiprotozoarios , Leishmania major , Leishmaniasis Cutánea , Leishmaniavirus , Animales , Antiprotozoarios/farmacología , Antiprotozoarios/uso terapéutico , Leishmania major/genética , Leishmaniasis Cutánea/parasitología , Leishmaniavirus/genética , Meglumina/uso terapéutico , Antimoniato de Meglumina/uso terapéutico , Ratones , Ratones Endogámicos BALB C , Reacción en Cadena de la PolimerasaRESUMEN
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.
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Epítopos Inmunodominantes/aislamiento & purificación , Leishmania infantum/inmunología , Leishmaniasis Visceral/diagnóstico , Proteómica/métodos , Secuencia de Aminoácidos , Antígenos de Protozoos/aislamiento & purificación , Western Blotting , Biología Computacional/métodos , Electroforesis en Gel Bidimensional , Chaperón BiP del Retículo Endoplásmico , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Epítopos/aislamiento & purificación , Humanos , Immunoblotting , Leishmaniasis Visceral/inmunología , Conformación Molecular , Estructura Secundaria de Proteína , Proteómica/normas , Proteínas Protozoarias/aislamiento & purificación , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Pruebas Serológicas/métodos , Pruebas Serológicas/normas , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
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.
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Antiprotozoarios/farmacología , Leishmania tropica/efectos de los fármacos , Antimoniato de Meglumina/farmacología , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Animales , Línea Celular , Cromatografía Liquida , Resistencia a Medicamentos , Leishmania tropica/aislamiento & purificación , Leishmania tropica/metabolismo , Ratones , Mitocondrias/efectos de los fármacos , Pruebas de Sensibilidad Parasitaria , Proteoma , Proteómica , Espectrometría de Masas en TándemRESUMEN
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.
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Leishmania tropica/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Pliegue de Proteína , Proteoma/metabolismo , Proteínas Portadoras , Chaperoninas , Cromatografía Liquida , Ciclo del Ácido Cítrico , Transporte de Electrón , Proteínas de Choque Térmico , Leishmania/metabolismo , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/aislamiento & purificación , Espectrometría de Masas en TándemRESUMEN
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.
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Variación Genética , Leishmania major/genética , Leishmaniasis Cutánea/diagnóstico , Leishmaniasis Cutánea/parasitología , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genética , Adolescente , Adulto , Anciano , Animales , Niño , Preescolar , ADN Protozoario/genética , Femenino , Humanos , Lactante , Irán/epidemiología , Leishmania major/clasificación , Leishmania major/enzimología , Leishmania major/aislamiento & purificación , Leishmaniasis Cutánea/epidemiología , Masculino , Persona de Mediana Edad , Filogenia , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Análisis de Secuencia de ADN , Piel/parasitología , Piel/patología , Adulto Joven , Zoonosis/parasitologíaRESUMEN
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.
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Enfermedades de los Perros/diagnóstico , Leishmania infantum/genética , Leishmaniasis Visceral/veterinaria , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Pruebas de Aglutinación , Animales , Anticuerpos Antiprotozoarios/sangre , ADN Intergénico/genética , ADN de Cinetoplasto/genética , ADN Protozoario/sangre , Exactitud de los Datos , Reservorios de Enfermedades , Enfermedades de los Perros/parasitología , Perros , Proteínas HSP70 de Choque Térmico/genética , Irán , Leishmania infantum/aislamiento & purificación , Leishmaniasis Visceral/diagnóstico , Metaloendopeptidasas/genética , Temperatura de TransiciónRESUMEN
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.
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ADN Protozoario/genética , Variación Genética/genética , Proteínas HSP70 de Choque Térmico/genética , Leishmania/genética , Filogenia , Proteínas Protozoarias/genética , Análisis de Secuencia de ADN , Animales , Humanos , Irán , Leishmania/aislamiento & purificación , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
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.
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Leishmania infantum/virología , Leishmania major/virología , Leishmania tropica/virología , Leishmaniavirus/genética , Leishmaniavirus/aislamiento & purificación , Animales , Análisis por Conglomerados , Gerbillinae , Humanos , Irán , Leishmania infantum/aislamiento & purificación , Leishmania major/aislamiento & purificación , Leishmania tropica/aislamiento & purificación , Leishmaniasis/parasitología , Leishmaniasis/veterinaria , Filogenia , ARN Polimerasa Dependiente del ARN/genética , Análisis de Secuencia de ADN , Homología de Secuencia , Proteínas Virales/genéticaRESUMEN
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.
RESUMEN
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.
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Leishmaniasis Visceral/epidemiología , Salud Rural , Anticuerpos Antiprotozoarios/sangre , Niño , Preescolar , Femenino , Política de Salud , Humanos , Irán/epidemiología , Leishmania infantum/inmunología , Leishmania infantum/aislamiento & purificación , Leishmania infantum/fisiología , Leishmaniasis Visceral/sangre , Leishmaniasis Visceral/parasitología , Masculino , Estudios SeroepidemiológicosRESUMEN
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.
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Anticuerpos Antiprotozoarios , Leishmania infantum , Leishmaniasis Visceral , Proteínas Protozoarias , Leishmania infantum/inmunología , Leishmaniasis Visceral/parasitología , Leishmaniasis Visceral/inmunología , Leishmaniasis Visceral/sangre , Humanos , Anticuerpos Antiprotozoarios/sangre , Proteínas Protozoarias/inmunología , Electroforesis en Gel Bidimensional , Antígenos de Protozoos/inmunología , Irán , ImmunoblottingRESUMEN
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.
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Clima , Irán/epidemiología , Humanos , Leishmaniasis/epidemiología , Leishmania/clasificación , Leishmania/aislamiento & purificación , Leishmania infantum/aislamiento & purificación , Sistemas de Información Geográfica , Temperatura , Leishmania tropica/aislamiento & purificación , Leishmania major/aislamiento & purificaciónRESUMEN
Background: Leishmaniasis is an important public health parasitic infection, which is endemic in many parts of the world, including Iran. We aimed to investigate genetic diversity and phylogenetic relationship among different Leishmania isolates using multi-locus sequence typing (MLST). Methods: Totally, 41 isolates collected either from patients referred to Leishmaniasis Diagnostics and Treatment Center at Tehran University of Medical Sciences, Tehran, Iran or from animals during 2019-2021, were subjected to the study. They included L. major and L. tropica from human, L. infantum from canine, and L. turanica from rodents from different endemic foci of Iran analyzed using MLST including gp63, g6pdh, lack, nagt, and hsp70 genes. Results: A total of 5010 bps was analyzed from each isolate. The three targets, nagt, lack, and g6pdh, generated better topology comparing to the other genes. In the 44 isolates, 22 haplotypes (STs) were identified. Leishmania tropica contained the highest number of haplotypes (n=12) comparing to L. major (n=8), L. infantum (n=1) and L. turanica (n=1). All five genomic loci caused separation of Iranian Leishmania species at the species level, indicating conservation of these genes in the Leishmania parasite. Conclusion: The highest number of haplotypes belonged to L. tropica, indicating that the genetic diversity of this species is higher than that of L. major. It was further confirmed that the MLST is a suitable method to examine genetic variation of Leishmania parasites with respect to evolutionary and epidemiological studies.
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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 (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.
Asunto(s)
Antimonio , Antiprotozoarios , Resistencia a Medicamentos , Glutatión , Glutatión/análogos & derivados , Leishmania tropica , Espermidina/análogos & derivados , Leishmania tropica/genética , Leishmania tropica/efectos de los fármacos , Resistencia a Medicamentos/genética , Animales , Antimonio/farmacología , Humanos , Antiprotozoarios/farmacología , Ratones , Glutatión/metabolismo , Línea Celular , Macrófagos/parasitología , Concentración 50 Inhibidora , Leishmaniasis Cutánea/parasitología , Leishmaniasis Cutánea/tratamiento farmacológico , Femenino , Adulto , Pruebas de Sensibilidad Parasitaria , Masculino , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
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.
Asunto(s)
Antiprotozoarios/farmacología , Resistencia a Medicamentos/genética , Leishmania tropica/efectos de los fármacos , Meglumina/farmacología , Compuestos Organometálicos/farmacología , Análisis del Polimorfismo de Longitud de Fragmentos Amplificados , Antimonio/farmacología , Línea Celular , Fragmentación del ADN , ADN Complementario/química , ADN Complementario/metabolismo , ADN Protozoario/química , ADN Protozoario/metabolismo , Genes Protozoarios , Concentración 50 Inhibidora , Leishmania tropica/genética , Antimoniato de Meglumina , Pruebas de Sensibilidad Parasitaria , ARN Protozoario/genética , ARN Protozoario/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
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.
Asunto(s)
Sistemas de Transporte de Aminoácidos/genética , Antimonio/farmacología , Antipruriginosos/farmacología , Resistencia a Medicamentos , Leishmania tropica/genética , Leishmaniasis Cutánea/parasitología , Proteínas Protozoarias/genética , Ubiquitina/genética , Sistemas de Transporte de Aminoácidos/metabolismo , Humanos , Leishmania tropica/efectos de los fármacos , Leishmania tropica/enzimología , Leishmania tropica/aislamiento & purificación , Proteínas Protozoarias/metabolismo , Ubiquitina/metabolismoRESUMEN
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.
Asunto(s)
Equinococosis Pulmonar , Cuidados Posoperatorios , Proteómica , Humanos , Cromatografía Liquida , Equinococosis Pulmonar/sangre , Equinococosis Pulmonar/cirugía , Pulmón , Espectrometría de Masas en Tándem , Factores de Transcripción , Biomarcadores/sangreRESUMEN
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.