Neutrophil extracellular traps formation: effect of Leishmania major promastigotes and salivary gland homogenates of Phlebotomus papatasi in human neutrophil culture.

BACKGROUND: Leishmaniasis as a neglected tropical disease (NTD) is caused by the inoculation of Leishmania parasites via the bite of phlebotomine sand flies. After an infected bite, a series of innate and adaptive immune responses occurs, among which neutrophils can be mentioned as the initiators. Among the multiple functions of these fighting cells, neutrophil extracellular traps (NETs) were studied in the presence of Leishmania major promastigotes and salivary gland homogenates (SGH) of Phlebotomus papatasi alone, and in combination to mimic natural conditions of transmission. MATERIAL & METHODS: The effect of L. major and SGH on NETs formation was studied in three different groups: neutrophils + SGH (NS), neutrophils + L. major (NL), neutrophils + L. major + SGH (NLS) along with negative and positive controls in 2, 4 and 6 h post-incubation. Different microscopic methods were used to visualize NETs comprising: fluorescence microscopy by Acridine Orange/ Ethidium Bromide staining, optical microscopy by Giemsa staining and scanning electron microscopy. In addition, the expression level of three different genes NE, MPO and MMP9 was evaluated by Real-Time PCR. RESULTS: All three microscopical methods revealed similar results, as in NS group, chromatin extrusion as a sign of NETosis, was not very evident in each three time points; but, in NL and especially NLS group, more NETosis was observed and the interaction between neutrophils and promastigotes in NL and also with saliva in NLS group, gradually increased over times. Real-time reveals that, the expression of MPO, NE and MMP9 genes increased during 2 and 4 h after exposure, and then decreased at 6 h in most groups. CONCLUSION: Hence, it was determined that the simultaneous presence of parasite and saliva in NLS group has a greater impact on the formation of NETs compared to NL and NS groups.

Inhibition of anti-inflammatory cytokines, IL-10 and TGF-ß, in Leishmania major infected macrophage by miRNAs: A new therapeutic modality against leishmaniasis.

Leishmania major (L. major) applies several mechanisms to escape the immune system. Interleukin-10 (IL-10) and Transforming Growth Factor (TGF-ß) downregulate nitric oxide synthase (iNOS) leading to the survival of Leishmania within macrophages. The miRNAs are known as the modulators of the immune system. The present study was conducted to assess the effect of synthetic miR-340 mimic on cytokines (IL-10 and TGF-ß1) involved in L. major infected macrophages. The miRNAs targeting of IL-10 and TGF-ß1 was predicted using bioinformatic tools. Relative expression of predicted miRNA, IL-10, and TGF-ß1 was measured by RT-qPCR before and after synthetic miRNA mimic transfection. Concentration of IL-10 and TGF-ß was measured in posttreatment condition using ELISA method. Also, infectivity was assessed by Giemsa staining. mmu-miR-340 received the highest score for targeting cytokines. The expression of miR-340 was downregulated in L. major infected macrophages. By contrast, expression of IL-10 and TGF-ß1 was upregulated in infected macrophages. After miRNA transfection, TGF-ß1 and IL-10 were both downregulated and interestingly, the combination of miR-340 and miR-27a had a stronger effect on the downregulation of target genes. This research revealed that transfection of infected macrophages with miR-340 alone or in combination with miR-27a mimic can reduce macrophage infectivity and might be introduced as a novel therapeutic agent for cutaneous leishmaniasis.

Recent researches in effective antileishmanial herbal compounds: narrative review.

Leishmaniasis are neglected diseases and a public health problem; they are caused by protozoan species belonging to the genus Leishmania and mostly influences the poor populations in many developing countries. The lack of effective medications, and an approved vaccine, high toxicity and life-threatening side effects and many cases of drug resistance reported in different countries have resulted in the necessity to discover new, efficient, inexpensive, and safe antileishmanial compounds with less or no toxicity. This increase in consumer demand of natural herbal-derived plant extracts as alternative medicines continues despite the low scientific information to establish their efficacy and safety profiles. Various studies have been conducted so far concerning the application of herbal medicines for the treatment of leishmaniasis, but research on relatively effective and low toxic substances is still needed. In this review, we have summarized recent developments and reported studies concerning about herbal and naturally derived therapeutics in the treatment of leishmaniasis, conducted by several researchers worldwide. Some of these medical herbs with promising results have undergone prospective clinical researches, but many others have either not yet been explored. Recent articles described these medical herbs and their active and important molecules, including quinones, phenolic derivatives, lignans, tannins, terpenes, and oxylipins. We searched ISI Web of Science, PubMed, SID, Scholar, Scopus, and Science Direct, and articles published up to 2019 were included. The keywords of leishmaniasis and some words associated with herbal medicines and natural products were used in our search. This review can serve as a quick reference database for researchers.

Candidate antigenic epitopes for vaccination and diagnosis strategies of Toxoplasma gondii infection: A review.

Toxoplasmosis caused by an obligatory intracellular protozoan parasite of Toxoplasma gondii threats a wide spectrum of human and animal hosts. It has been shown that the intensity of the disease in humans depends on the host's immune responses. Immunological investigations on whole protein molecules of T. gondii have shown that these antigens are not fully responsible for the immune response, which leads to a decrease in specificity and affinity of the antigen (epitope)-antibody (paratope) binding. Currently, epitopes have shown promising entities to stimulate B, T, cytotoxic T lymphocyte, and NK cells resulting in enhancement of protective immunity against toxoplasmosis patients. Thus, the accurate designing, prediction, and conducting of antigenic epitopes of T. gondii (with linear and/or spatial structures (can augment our understanding about development of new serological diagnostic kits and vaccines. The current review provides an update on the latest advances of current epitopes described against toxoplasmosis including B cell/T cell epitopes, antigen types, parasite strains, epitope sequences, assay settings (in vitro and/or in vivo), and target strategy. Present results disclosed that the designing of effective multiepitopes of T. gondii by in silico modeling and immunoinformatics tools can strengthen our knowledge about triggering of epitope-based vaccine/diagnosis strategies in future perspectives.

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.

Syphacia obvelata: A New Hope to Induction of Intestinal Immunological Tolerance in C57BL/6 Mice.

The ability of nematodes to manipulate the immune system of their host towards a Th2 and T regulatory responses has been proposed to suppress the inflammatory response. Clinical trials have proposed a useful effect of helminth infections on improvement of inflammatory disorders. In this study, we investigated the immunomodulatory effect of Syphacia obvelata infection to induce intestinal tolerance in C57BL/6 mice. Mice were infected through the cagemates with self-infected BALB/c mice. Four weeks post-infection, expression levels of IFN-γ, TNF-α, IL-17, and IL-10 were assessed in the supernatant of mesenteric lymph node (MLN) culture. Foxp3+Treg were measured in MLN cells by flow cytometry. In the S. obvelata-infected group, the percentage of Tregs (5.2±0.4) was significantly higher than the control (3.6±0.5) (P<0.05). The levels of IL-10 (55.3±2.2 vs 35.2±3.2), IL-17 (52.9±3.8 vs 41±1.8), IFN-γ (44.8±4.8 vs 22.3±2.3) and TNF-α (71.1±5.8 vs 60.1±3.3) were significantly increased in infected mice compared to the control group (P<0.05). The above results showed the potential effects of S. obvelata to induce intestinal tolerance. Therefore, it seems that S. obvelata may increase the immunological suppressive function in the intestinal tract.

Effect of simultaneous and sequential use of TGF-ß1 and TGF-ß3 with FGF-2 on teno/ligamentogenic differentiation of periodontal ligament stem cells.

OBJECTIVE: The periodontal ligament is a crucial part of the periodontium, and its regeneration is challenging. This study compares the effect of simultaneous and sequential use of FGF-2 and TGF-ß1 with FGF-2 and TGF-ß3 on the periodontal ligament stem cells (PDLSCs) teno/ligamentogenic differentiation. DESIGN: This study comprises ten different groups. A control group with only PDLSCs; FGF-2 group containing PDLSCs with a medium culture supplemented with FGF-2 (50 ng/mL). In other experimental groups, different concentrations (5 ng/mL or 10 ng/mL) of TGF-ß1&-ß3 simultaneously or sequentially were combined with FGF-2 on the cultured PDLSCs. TGF-ß was added to the medium after day 3 in the sequential groups. Methyl Thiazolyl Tetrazolium (MTT) assay on days 3, 5, and 7 and Quantitative Real-time Polymerase Chain Reaction (RT-qPCR) analysis after day 7 were conducted to investigate PLAP1, SCX, and COL3A1, RUNX2 genes. All experiments were conducted in a triplicate. The One-way and Two-way ANOVA with Tukey post hoc were utilized to analyze the results of the MTT and RT-qPCR tests, respectively. A p-value less than 0.05 is considered significant. RESULTS: The proliferation of cells on days 3, 5, and 7 was not significantly different among different experimental groups (P > 0.05). A higher expression of the PLAP1, SCX, and COL3A1 have been seen in groups with sequential use of growth factors; among these groups, the group using 5 ng/mL of TGF-ß3 led other groups with the most amount of significant upregulation in PLAP1(17.69 ± 1.11 fold; P < 0.0001), SCX (5.71 ± 0.38 fold; P < 0.0001), and COL1A3 (6.35 ± 0.39 fold; P < 0.0001) expression, compared to the control group. The expression of the RUNX2 decreased in all groups compared to the control group; this reduction was more in groups with sequential use of growth factors. CONCLUSION: The sequential use of growth factors can be more effective than simultaneous use in teno/ligamentogenic differentiation of PDLSCs. Moreover, treatment with 5 ng/mL TGF-ß3 after FGF-2 was more effective than TGF-ß1.

The promising prospect of human hair follicle regeneration in the shadow of new tissue engineering strategies.

Hair loss disorder (alopecia) affects numerous people around the world. The low effectiveness and numerous side effects of common treatments have prompted researchers to investigate alternative and effective solutions. Hair follicle (HF) bioengineering is the knowledge of using hair-inductive (trichogenic) cells. Most bioengineering-based approaches focus on regenerating folliculogenesis through manipulation of regulators of physical/molecular properties in the HF niche. Despite the high potential of cell therapy, no cell product has been produced for effective treatment in the field of hair regeneration. This problem shows the challenges in the functionality of cultured human hair cells. To achieve this goal, research and development of new and practical approaches, technologies and biomaterials are needed. Based on recent advances in the field, this review evaluates emerging HF bioengineering strategies and the future prospects for the field of tissue engineering and successful HF regeneration.

Recent approaches in regenerative medicine in the fight against neurodegenerative disease.

Neurodegenerative diseases arise due to slow and gradual loss of structure and/or function of neurons and glial cells and cause different degrees of loss of cognition abilities and sensation. The little success in developing effective treatments imposes a high and regressive economic impact on society, patients and their families. In recent years, regenerative medicine has provided a great opportunity to research new innovative strategies with strong potential to treatleva these diseases. These effects are due to the ability of suitable cells and biomaterials to regenerate damaged nerves with differentiated cells, creating an appropriate environment for recovering or preserving existing healthy neurons and glial cells from destruction and damage. Ultimately, a better understanding and thus a further investigation of stem cell technology, tissue engineering, gene therapy, and exosomes allows progress towards practical and effective treatments for neurodegenerative diseases. Therefore, in this review, advances currently being developed in regenerative medicine using animal models and human clinical trials in neurological disorders are summarized.

The Current Status and Future Direction of Extracellular Nano-vesicles in the Alleviation of Skin Disorders.

Exosomes are extracellular vesicles (EVs) that originate from endocytic membranes. The transfer of biomolecules and biological compounds such as enzymes, proteins, RNA, lipids, and cellular waste disposal through exosomes plays an essential function in cell-cell communication and regulation of pathological and physiological processes in skin disease. The skin is one of the vital organs that makes up about 8% of the total body mass. This organ consists of three layers, epidermis, dermis, and hypodermis that cover the outer surface of the body. Heterogeneity and endogeneity of exosomes is an advantage that distinguishes them from nanoparticles and liposomes and leads to their widespread usage in the remedy of dermal diseases. The biocompatible nature of these extracellular vesicles has attracted the attention of many health researchers. In this review article, we will first discuss the biogenesis of exosomes, their contents, separation methods, and the advantages and disadvantages of exosomes. Then we will highlight recent developments related to the therapeutic applications of exosomes in the treatment of common skin disorders like atopic dermatitis, alopecia, epidermolysis bullosa, keloid, melanoma, psoriasis, and systemic sclerosis.

Detection and phylogenetic analysis of kinetoplast DNA of Leishmania infantum infected humans, domestic dogs and sandflies in Northwest Iran.

Leishmaniasis refers to a disease with a wide range of manifestations; and there are three main forms of disease, cutaneous, mucocutaneous, and visceral. Leishmaniasis is one of the diseases with a protozoan agent which is vector-borne. Visceral leishmaniasis (VL) is the most severe form that can be fiercely life-threatening if left untreated. VL can be caused by members of Leishmania donovani complex, in Iran, Leishmania infantum is considered the primary causative agent of VL, resulting in a zoonotic form of VL. The two main goals of our work, which followed our prior sero-epidemiological and entomological survey, were to characterize and conduct a phylogenetic analysis of the Leishmania species that infect people, dogs, and sandflies. The samples were collected throughout 2017, from January to December, so blood samples were collected from humans and dogs, while sandfly samples were collected with sticky traps. DNA extracted from all seropositive samples of humans and dogs, 10% of sero-negative human samples, and all collected sandflies were subjected to kDNA-nested-PCR for tracing parasites. A total of 30 samples, including 20 human samples, 8 dog samples, and 2 sandfly samples, were found positive for the kDNA gene of L. infantum. Sequences were evaluated to study the genetic diversity among the six discovered L. infantum. Based on kDNA, the phylogenetic study of L. infantum demonstrated a high level of genetic variety and a relationship between the host, the parasite's geographic origin, and its genetic diversity.

Therapeutic effect of trace elements on multiple myeloma and mechanisms of cancer process.

In the human body, trace elements and other micronutrients play a vital role in growth, health and immune system function. The trace elements are Iron, Manganese, Copper, Iodine, Zinc, Cobalt, Fluoride, and Selenium. Estimating the serum levels of trace elements in hematologic malignancy patients can determine the severity of the tumor. Multiple myeloma (MM) is a hematopoietic malignancy and is characterized by plasma cell clonal expansion in bone marrow. Despite the advances in treatment methods, myeloma remains largely incurable. In addition to conventional medicine, treatment is moving toward less expensive noninvasive alternatives. One of the alternative treatments is the use of dietary supplements. In this review, we focused on the effect of three trace elements including iron, zinc and selenium on important mechanisms such as the immune system, oxidative and antioxidant factors and cell cycle. Using some trace minerals in combination with approved drugs can increase patients' recovery speed. Trace elements can be used as not only a preventive but also a therapeutic tool, especially in reducing inflammation in hematological cancers such as multiple myeloma. We hope that the prospect of the correct use of trace element supplements in the future could be promising for the treatment of diseases.

Identification and genotyping of Acanthamoeba spp. in the water resources of western Iran.

Background: Acanthamoeba spp. is opportunistic amoeba that resides in water, soil, and air. Some pathogenic genotypes of the genus of Acanthamoeba can cause granulomatous amoebic encephalitis (GAE) in people with a defective immune system. The parasite can also cause Acanthamoeba keratitis (AK) among contact lens users. This study was conducted to isolate and identify the Acanthamoeba genotypes in water resources in Lorestan province, western Iran. Methods: Collected 72 water samples from surface and groundwater (springs and aqueducts) in Lorestan province. Samples were filtered and cultured in non-nutrient 1.5% agar medium covered with Escherichia coli (E. coli) at 25 °C. DNA extraction was done and the PCR reaction was performed to detect the Acanthamoeba spp. The positive PCR products were sequenced to determine the genotypes of Acanthamoeba. Results: Out of 72 examined water samples, 23.61% were positive for Acanthamoeba sp. by PCR. From PCR-positive samples, 8 (47.05%) samples were T4 genotypes and others were other Acanthamoeba genotypes (T1-T23). Therefore, approximately half of the genotypes belong to the pathogenic T4 genotype. Conclusions: The water examined samples in western provinces of Iran have the potential risk factor for public health. Therefore, the efforts of healthcare providers are needed to identify, train, and prevention from human infections.

TGF-ß Targeted by miR-27a Modulates Anti-Parasite Responses of Immune System.

Background: Immune cells and their secreted cytokines are known as the first barrier against pathogens. Leishmania major as an intracellular protozoan produces anti-inflammatory cytokines that lead to proliferation and survival of the parasite in the macrophages. miRNAs are small non-coding RNA molecules that regulate mRNAs expression. We aimed to investigate the relationship between the expression of TGF-ß and a bioinformatically candidate miRNA, in leishmaniasis as a model of TGF-ß overexpression. Methods: The miRNAs that target TGF-ß -3'UTR were predicted and scored by bioinformatic tools. After cloning of TGF-ß-3'UTR in psi-CHECK ™- 2 vector, targeting validation was confirmed using Luciferase assay. After miRNA mimic transfection, the expression of miR-27a, TGF-ß, as well as Nitric Oxide concentration was evaluated. Results: miR-27a received the highest score for targeting TGF-ß in bioinformatic predictions. Luciferase assay confirmed that miR-27a is targeting TGF-ß-3'UTR, since miR-27a transfection decreased the luciferase activity. After miRNA transfection, TGF-ß expression and Nitric Oxide concentration were declined in L. major infected macrophages. Conclusion: Bioinformatic prediction, luciferase assay, and miRNA transfection results showed that miR-27a targets TGF-ß. Since miRNA and cytokine-base therapies are developing in infectious diseases, finding and validating miRNAs targeting regulatory cytokines can be a novel strategy for controlling and treating leishmaniasis.

Thermotolerant Acanthamoeba spp. isolated from therapeutic hot springs in Northwestern Iran.

This study was conducted to address the distribution of Acanthamoeba genotypes in therapeutic hot springs in Iran. Sixty water and sediment samples were collected from bicarbonate, sulphur, and sodium chloride thermal springs in the northwest. All hot springs examined are used mainly for health purposes in Iran. Acanthamoeba were identified by both morphology and PCR (polymerase chain reaction). Genotype identification was based on the sequencing of a highly variable and informative region of Diagnostic Fragment 3 (stem 29-1 of 18S rRNA gene) within Acanthamoeba-specific amplimer (ASA.S1). Twenty percent of hot springs were contaminated with thermotolerant Acanthamoeba belonging to the potentially pathogenic T4 and T3 genotypes. A high number (91.7%) of strains showed growth at 37 °C, and eight isolates showed growth at 42 °C. A single isolate (HSNW2) was detected in waters at 70 °C. The presence of thermotolerant Acanthamoeba highlights a risk factor for susceptible individuals, as Acanthamoeba-related keratitis continues to rise in Iran. Periodic surveillance of thermal waters as well as improved filtration and disinfection is recommended to prevent disease related to pathogenic Acanthamoeba. This is the first comprehensive molecular study of Acanthamoeba genotypes in hot springs in Iran and the first to report the occurrence of the T3 genotype (corresponding to Acanthamoeba griffini) in thermal water sources in this country.

Isolation of Acanthamoeba species in surface waters of Gilan province-north of Iran.

We analyzed water samples to determine the prevalence of free-living Acanthamoeba in water sources from Gilan, greater area, Iran. A total of 27 surface water samples were collected from environmental sources, including natural (rivers, lakes, springs, and lagoon) and freshwater source. The samples were filtrated and transferred to non-nutrient agar plates seeded with Escherichia coli and incubated for 2 to 7 days at 30°C or 42°C. The plates were examined by microscopy to morphologically identify Acanthamoeba species. Following DNA extraction, PCR was used to confirm the microscopically identification. A total of 19 out of 27 samples (70.3%) were positive for Acanthamoeba species based on the morphological criteria, and 14 (73.7%) were confirmed by PCR method. The high frequency of Acanthamoeba spp. in different environmental water sources of Gilan is an alert for the public health related to water sources in Iran.

Molecular identification of Hartmannella vermiformis and Vannella persistens from man-made recreational water environments, Tehran, Iran.

A survey was conducted on man-made recreational water located in different regions of Tehran, Iran to detect the free-living amoebae present in ponds and fountains of parks and squares. Fifty water samples from 22 municipal districts of Tehran were screened for free-living amoebae and identified by morphological characters and polymerase chain reaction amplification. Amoebae detected were identified as Hartmannella vermiformis (12 %) and Vannella persistens (4 %), which are the first reports of these two amoebas in recreational water environments of Iran. Since, H. vermiformis, which is highly similar to strains serving as hosts for Legionella pneumophila, is a common component of the microbial community in fresh surface water. Although Vannella spp. is not proved to be pathogenic itself, they are capable of harboring pathogenic intracellular organisms. Due to some reports related to pathogenicity of these amoebas, the particular hazard related to these microorganisms should be taken into account in the encounter with drinking and washing in these waters. We recommend control strategies based on physical removal rather than on disinfection to be adopted where necessary.

The effect of extracellular matrix remodeling on material-based strategies for bone regeneration: Review article.

Bone defects may cause by a number of acquired or inherited disorders. Tissue engineering strategies aim to induce functional bone regeneration through incorporating biomaterials and cells, which can potentially provide an efficient and personalized treatment option with reduced risk of rejection. Designing the appropriate scaffold for each tissue is critical because of the microenvironment where cell growth can occur. Various types of natural and synthetic polymers were studied in combination with active ceramic and metallic materials to form osteoconductive scaffolds. The purpose of producing composite scaffolds was to obtain a supporting structure with appropriate mechanical and surface properties to mimic the bone extracellular matrix (ECM). The ECM is a dynamic bio-environment that continuously undergoes remodeling to control tissue homeostasis. This process is mediated by specific proteases such as metalloproteinases (MMPs) that play an essential role in ECM degradation. The ECM is involved in regulating cell adhesion, proliferation, differentiation, and finally, the functional properties of the mature bone. Many substances have different effects on ECM, which in turn can be effective in bone regeneration. Imitation of ECM is one of the promising ways in designing materials for the bone regeneration. In this review, we investigated the effect of ECM remodeling on material-based strategies for bone regeneration.

Potentially pathogenic free-living amoebae isolated from hospital wards with immunodeficient patients in Tehran, Iran.

This study investigated the occurrence of free-living amoebae (FLA) in immunodeficiency wards of hospitals in Tehran, Iran. A total of 70 dust and biofilm samples from wards serving transplant, pediatric (malignancies), HIV, leukemia and oncology patients of five university hospitals were collected and examined for the presence of FLA using culturing and molecular approaches. Based on the morphology of the amoebae in plate cultures, primer sets were applied for molecular identification of Acanthamoeba, vahlkampfiid amoebae and Hartmannella. Out of 70 samples, 37 (52.9%) were positive for FLA. Acanthamoeba belonged to the T4 genotype was the most prevalent isolate. Presence of the T4 genotype on medical instruments, including an oxygen mask in an isolation room of an immunodeficiency pediatric ward, should be of concern for health authorities. Acanthamoeba T5 genotypes, Hartmannella vermiformis, and Vahlkampfia avara were also present. These results highlight a clear need for greater attention to improved disinfection, especially where susceptible patients, such as those who are immune-suppressed, are served. To our knowledge, this is the first report of these FLA in immunodeficiency wards in Iran, and also the first to identify Acanthamoeba T5, Hartmannella, and Vahlkampfia in moist habitats, such as biofilms, in this country.

Comparison of Three Commonly Used Genetic Markers for Detection of Leishmania Major: An Experimental Study.

BACKGROUND: Leishmaniasis is a vector-borne disease caused by an intracellular protozoan parasite called Leishmania spp. Different species produce different clinical outcomes; the majority of cases are cutaneous forms. Leishmania major is one of the main causative agents of cutaneous leishmaniasis (CL). Various methods are being using to diagnose CL, including microscopic examination, culture, and molecular detection of the parasite genome. METHOD: In the current study, we tried to compare three common molecular markers, including Kinetoplast DNA (kDNA), Cytochrome b (Cyt b), and Internal transcribed space 1 (ITS1), for the detection of Leishmania major. After cultivation of standard strain of L. major MHOM/IR/75/ER in RPMI 1640, certain number of promastigotes was subjected to DNA extraction and different PCR reactions. RESULTS: The lowest number of the parasite (5 promastigotes) can be detected by kDNA-PCR, followed by Cyt b-PCR (10 promastigotes), and ITS1-PCR (50 promastigotes). CONCLUSION: In conclusion, kDNA-PCR was the most sensitive marker and may provide more reliable data in the initial screening, especially in false-negative results provided by parasitological methods due to the low number of parasites.