Molecular identification of Acanthamoeba genotypes isolated from oral cavity of heart transplant patients in Iran.

OBJECTIVE: Heart transplant is one of the accepted treatments for some patients with advanced heart failure. Of note, transplant surgeries may cause different infections and complications for patients during the post-transplant period. A wide variety of opportunistic organisms caused these infections including bacteria, fungi, viruses, and protozoa particularly Free-living amoebae (FLA). This study aims to study the presence of pathogenic FLA from the oral cavity of post-heart transplant recipients. METHODS: Throat swabs were collected from 80 patients who underwent post-heart transplant surgery. All swabs were immediately cultured in non-nutrient agar (2%). PCR and sequencing of 18S rRNA gene (DF3 region) of Acanthamoeba isolates were performed using genus-specific primers. Genetic associations among sequenced genotypes inferred by the 18S rRNA gene obtained by MEGA X and a phylogenetic tree were constructed using the maximum likelihood algorithm and Kimura 2-parameter model. RESULTS: Out of 80 samples collected from post-heart transplant patients, six (7.5%) samples showed positive outgrowth of Acanthamoeba based on the page key and sequencing of the DF3 region. Sequence similarity of ASA1 by basic local alignment search tool(n) showed that five isolates (ANHT1, ANHT2, ANHT3, ANHT4, and ANHT5) belonged to Acanthamoeba T5 genotype corresponding to A. lenticulata and one strain (ANHT6) belonged to the T4 genotype. CONCLUSION: To the best of our knowledge for the first time, a comprehensive study of Acanthamoeba genotypes isolated from throat samples of heart transplant recipients is described. Heart transplantation patients can be colonized by FLA and are therefore at risk of developing an invasive infection. Physicians' awareness of central nervous system infections related to FLAs and preventive and control measures of patients with compromised immune status due to heart transplant surgery are of utmost importance.

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.

Isolation of Balamuthia mandrillaris from soil samples in North-Western Iran.

Balamuthia mandrillaris is an opportunistic free-living amoebae (FLA) which has been reported as the causative agent of the fatal Balamuthia amoebic encephalitis (BAE). Currently, the transmission dynamics of this pathogen remain poorly understood although the organism has been associated with soils, and thus, soil exposure has been identified as a risk factor for this pathogenic amoeba. Nevertheless, environmental isolation of B. mandrillaris is a rare event and strains of this amoebic species have been isolated from soil and dust sources only in seven previous reports (Iran, Mexico, USA, Peru, Costa Rica, Guinea Bissau, and Jamaica). In Iran, a previous study reported the isolation of B. mandrillaris from dust collected in the city of Tehran and free of known infectious involvement. Therefore, in this work, 55 soil samples collected from public and recreational areas of East Azerbaijan, North-Western Iran, were investigated for the presence of this pathogen. Samples were cultured in 2% non-nutrient agar plates and were monitored for the presence of B. mandrillaris-like trophozoites and/or cysts. Those samples that were positive for these amoebae by microscopic criteria were then confirmed by PCR amplification and DNA sequencing of the mitochondrial 16S ribosomal DNA (rDNA) of B. mandrillaris. The obtained results revealed the presence of this emerging pathogen in 5 of 55 samples included in the study. Homology analysis of the obtained DNA sequences revealed high similarity with previously isolated strains from clinical and environmental sources. To the best of our knowledge, this is the first report on the isolation of B. mandrillaris from soil sources in Iran related to human activity and the second time that this pathogen is isolated from the environment in this country.

Ophthalmology hospital wards contamination to pathogenic free living Amoebae in Iran.

The present study was conducted to determine the occurrence of potentially pathogenic free-living amoeba in ophthalmology wards in reference hospitals in Iran. Since an increasing number of Acanthamoeba Keratitis cases after eye surgery and eye trauma have been recently observed in this country, it could be possible that the disinfection procedures undertaken in the clinical setting may not have a good hygiene and disinfection procedures, hence the aim of this study. Therefore, 42 dust and biofilm samples were collected from different areas of ophthalmology wards and checked for the presence of FLA using morphological criteria, PCR based analysis and DNA sequencing. Of the 42 samples from dust and biofilm sources, 18(42.86%) isolates were found to contain FLA and 12(92.3%) isolates belonged to Acanthamoeba T4 genotype. Isolation of the pathogenic genotype T4 from medical instruments, including slit lamp in corneal wards, may be a threat for patients undergoing eye surgery in these wards. Other FLA isolated in this study included Acanthamoeba genotype T5, Vahlkampfia sp, Naegleria australiensis, Vermamoeba vermiformis and Echinamoeba exudans. To our knowledge, this is the first report of the presence of potentially pathogenic FLA in ophthalmology wards in Iran. Improved disinfection methods and monitoring of hospitals ward are thus necessary in this area in order to minimize the risk of infection in patients.

Molecular Characterization of Pathogenic Acanthamoeba Isolated from Drinking and Recreational water in East Azerbaijan, Northwest Iran.

Acanthamoeba- related infections, such as amoebic keratitis and granulomatous amoebic encephalitis, can develop in high-risk population through contaminated water sources. Thus, surveying water resources, particularly those available for human use, is of the utmost importance. In the present study, 67 water samples were collected from water resources in East Azerbaijan, a province in northwestern Iran. Samples were cultured on enriched non-nutrient agar plates, and sequencing-based approaches were used for genotyping. The pathogenic potential of the isolates was determined using thermo- and osmo-tolerance tests. Acanthamoeba were detected in 17 (25.4%) of the 67 collected samples. Sequencing analysis revealed that the isolates belonged to the T3 (23.52%), mixed T3/T4 (5.88%), T4 (58.82%), T5 (5.88%), and T13 (5.88%) genotypes. Through thermo- and osmo-tolerance tests, 88.23% of isolates were resistant to 37 °C, 40 °C temperature, and 0.5 M and 1 M osmolarity; thus, these isolates had the potential for pathogenicity. These findings point toa serious public health concern in the studied region. This study is the first to report Acanthamoeba isolated from drinking and recreational water sources in East Azerbaijan and Acanthamoeba T13 isolated from tap water in Iran.

Optimization of fluoride-containing bioactive glasses as a novel scolicidal agent adjunct to hydatid surgery.

We have presented a novel scolicidal agent made from fluoride-containing bioactive glass (FBG). The samples were characterized by XRD and the ion release behavior of the samples was evaluated at 37°C. The scolicidal activity, cellular response and antibacterial activity and biocompatibility of the different concentrations of the FBG (ranging from 0.5 to 2 mg ml(-1)) were evaluated in vitro and in vivo. The results showed an easy diffusion of the fluoride through the glass matrix in an F concentration-dependent manner. The FBG showed an intensive scolicidal property, so that toxicity effect begun from 5 min and the samples with 20, 10, 5 and 0% fluoride showed 98±2, 93±5.8, 76.2±6 and 5.8±1.7% scolicidal activity, respectively, after 8h exposing time. Our data revealed that scolicidal activity of the FBG increased with the increase of F ratio and concentrations of the bioactive glass. On the other hand, all the synthesized FBG samples found to be cyto-biocompatible when tested in vitro (MTT and LDH assays) and in vivo (no significant infiltration of inflammatory cells compared with control, p>0.05), and antibacterial against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. The results demonstrated that the synthesized FBG might have a potential application in prevention of post-surgery infections, especially hydatidosis.

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.