Dimedone nanoparticle as a promising approach against toxoplasmosis: In vitro and in vivo evaluation.

Toxoplasma gondii, an intracellular parasite, has shown drug resistance and therapeutic failure in recent years. Dimedone (DIM) has been introduced as a new chemical compound with anti-bacterial and anti-cancer properties. The aim of this study was to investigate the potential protective role of DIM nanoparticles in an animal model of toxoplasmosis. Cytotoxicity of DIM on Vero cell line assessed using MTT, and the effect of DIM on Toxoplasma gondii was evaluated by counting the number of parasites compared to the control group in vitro. The rate of pathogenesis and virulence of the parasite was checked on the liver cells of the animal model using hematoxylin-eosin staining. Furthermore, various parameters indicating oxidative stress were compared in mouse liver tissue in different groups. The release of the nanoparticle form was significantly longer than the free drugs. The IC50 of Nano-DIM was 60 µM and the reduction of intracellular parasite proliferation in the group Nano-DIM and Nano-PYR (Nano-primethamine) was significantly lower than the free drugs in vitro. Histopathology examination in the groups treated with dimedone nanomedicine showed that the degree of disintegration of the epithelium of the central vein of the liver and infiltration and vacuolization of liver cells were lower compared to the toxoplasmosis group. Additionally, the level of some oxidative stress indicators was observed to be lower in the nano-treated groups compared to other groups. The results of this study showed DIM can be used as a promising compound for anti-T. gondii activity and can prevent the proliferation of it in cells.

Design of highly sensitive nano-biosensor for diagnosis of hydatid cyst based on gold nanoparticles.

Cystic echinococcosis, a zoonotic parasitic infection, is a major public health and economic concern, with worldwide distribution. The development of sensitive diagnostic methods for hydatid disease is important. We designed a highly sensitive nano-biosensor for the diagnosis of hydatid cyst based on gold nanoparticles (AuNPs). AuNPs were synthesized. Echinococcus granulosus antigen was coated on the ELISA microwells. Then, the E. granulosus IgG antibody was added to the microwells. After incubation and washing, the Ag-Ab complex was incubated with a human IgG HRP​-conjugated antibody. Then, the synthesized AuNPs and tetramethylbenzidine (TMB), as a chromogenic substrate of HRP, were added to the reaction. Finally, the absorption rate was measured by spectrophotometry. The results showed that the enzyme peroxide and TMB change the color of the reaction from red to yellow by oxidizing AuNPs. The sensitivity and specificity of the designed method were investigated. The linear equation and regeneration of nanobiosensor designed for red color Y = 0.0312X + 0.649, R2 9962 and for yellow color Y = 0.013X + 0.398, R2 9851 were determined. The limit of detection of the designed nanobiosensor was 0.001 µg mL-1. The results confirmed that the designed nanobiosensor was completely specific for the detection of E. granulosus antibody.

Genotyping of Fresh and Parafinized Human Hydatid Cysts Using nad1 and cox1 Genes in Hamadan Province, West of Iran.

BACKGROUND: Hydatidosis is a cosmopolitan zoonotic infection and Hamadan Province in the west of Iran is one of the most important foci of human hydatidosis in Iran. The aim of the current study was the genetic characterization of hydatid cysts operated from humans in Hamadan Province. METHODS: Seventy-two hydatid cysts samples including 50 paraffinized and 22 fresh human hydatid cysts collected from different hospitals in Hamadan Province, western Iran. The cysts' DNA genome was extracted by kit and PCR was performed for amplifying the fragments of 400 and 450bp for nad1 and cox1 mitochondrial genes, respectively. Genotype diversity and sequence variations of the cysts' isolates were studied by related software. RESULTS: DNA from all (100%) paraffinized and fresh hydatid cysts samples extracted successfully. All paraffinized and fresh hydatid cysts samples were amplified by PCR assay using nad1gene, however, only 18 and 8 samples from paraffinized and fresh hydatid cyst samples was amplified using cox1 gene, respectively. The sequences analysis indicated that, 98.61% the Echinococcus granulosus samples were belong to the genotype G1 and 1.39% were G3 genotype. CONCLUSION: Genotypes of E. granulosus in human samples in Hamadan Province are G1 and G3 and these findings are proved by phylogenic analysis.

Genotyping of Echinococcus granulosus isolates from livestock based on mitochondrial cox1 gene, in the Markazi province, Iran.

Hydatidosisis a parasitic disease caused by the larval stage of Echinococcus granulosus with different genotypes, and major complications in vital organs such as liver, lungs and, brain. Also, this parasite can infect animals and cause economic damages. Recently, some investigations indicated that the genetic variation of the parasite affects the antigenic, immunogenic and pathogenic features. Therefore, present study conducted to genotyping of the E. granulosus larva based on mitochondrial cox1 gene in livestock in the endemic areas of Markazi province, Iran. In this study, 49 hydatid cysts samples collected from 36 sheep, 11 goats and 2 cattle from different slaughterhouses of Markazi province in central part of Iran, 2017. The mitochondrial cox1 gene was amplified and genotyping were accomplished using sequence analysis. The sequencing analysis indicated that the main genotype G1 (61%) and G3 (37%) were identified. Also, one of the samples shows similarity with the G2 (2%) genotype. The results showed the statistically significant differences between the genotypes in different livestock (P < 0.05). This study indicated that the main genotypes of E. granulosus in Markazi province are G1 and G3 which are related to dog/sheep strain. Therefore, parasite control in dogs and sheep can reduce the risk of transmission of infection to humans.

Giardia lamblia assemblages A and B isolated from symptomatic and asymptomatic persons in Hamadan, west of Iran.

Giardia is a very abundant organism bringing about diarrhoea in human beings. The focus of this analysis was the detection of Giardia lamblia assemblages in human stool specimens in Hamadan, west of Iran, as well as the association between obtained assemblages and clinical symptoms. Faecal samples of 4066 individuals admitted to the medical and health care facilities in Hamadan were inspected microscopically for the existence of Giardia cysts/trophozoites, and the clinical symptoms of the patients were recorded. The DNA of positive samples was isolated from and the nucleotide sequences of both glutamate dehydrogenase (gdh) (n = 15) and triose phosphate isomerase (tpi) (n = 8) genes were analyzed. In direct microscopy, a total of sixty-four samples (1.6%), were considered as positive for G. lamblia cysts or trophozoites. The sequence analysis showed that 18 out of 23 sequenced isolates (78.2%) were assemblage A and 5 (21.7%) were assemblage B. Clinical symptoms were observed in 44.4% and 40% of patients with assemblages A and B, respectively. Overall, the predominant assemblage A detected in the tested samples along with bioinformatics analysis suggest a potential zoonotic transmission in the region of the study. Although advanced analyses are necessary to understand the foundation and path of the infection, it seems that more sanitary regulations regarding contact with livestock and pet animals are essential.

Echinococcus granulosus sensu stricto in Livestock and Human in Hamadan, Western Iran.

BACKGROUND: Cystic echinococcosis, a major public health and economic concern, is a zoonotic helminth infection with worldwide distribution. This study was conducted to investigate the genetic characteristics of hydatid cysts isolated from human and livestock in Hamadan region, western Iran during 2016-2017. METHODS: Ten human hydatid cysts and 40 animal hydatid cysts including 32 sheep, 5 cattle and 3 goats were genotyped by PCR amplification of two mitochondrial genes, cox1 and nad1. Genetic identification of the isolates was performed by using bioinformatics software and mtDNA nucleotide sequences of the parasite, available in GenBank database. RESULTS: The PCR amplification was successfully carried out on 50 hydatid cyst isolates and then the nucleotide sequencing was conducted. The sequence analysis of the samples found that the isolates belonged to E. granulosus sensu stricto including G1 (42/50, 84%), G2 (4/50, 8%) and G3 (4/50, 8%) genotype. The G1 genotype was detected in human (8/10, 80%), sheep (26/32, 81%), cattle (5/5, 100%) and goat (3/3, 100%) hydatid cysts. The G2 and G3 genotypes were found only in sheep and human isolates. Alignment analysis of the cox1 and nad1 gene sequences revealed thirteen and ten sequence types, respectively. CONCLUSION: G1 was the prevailing genotype of E. granulosus in the area and dog-sheep transmission cycle should be considered when implementing hydatidosis control programs. In addition, high genetic diversity was detected among the hydatid cyst isolates.

Genetic Identification of Echinococcus granulosus Isolates in Hamadan, Western Iran.

BACKGROUND: Cystic echinococcosis is a zoonotic infection and considered as a major economic and public health concern worldwide. This research was conducted to determine genotypic characteristics of livestock and human hydatid cyst isolates from Hamadan area, western Iran. METHODS: Sampling was conducted in Hamadan industrial slaughterhouse and Beast Hospital of Hamadan City, western Iran, from 2015 to 2016. Overall, 74 livestock isolates including 69 sheep, 3 cattle and 2 goats and 9 human hydatid cysts were genotyped by PCR amplification of the rDNA ITS1 region and followed restriction fragment length polymorphism (RFLP) analysis with four restriction endonuclease enzymes, RsaI, HpaII, AluI, and TaqI, and sequencing. RESULTS: The PCR amplicon size of each isolate was approximately 1 kb which was the same with that of sheep strain. According to the RFLP patterns, the isolates belonged to a single species, E. granulosus sensu stricto (G1-G3 complex). Furthermore, sequencing of representative amplicons confirmed that the RFLP-genotyped isolates corresponded to E. granulosus sensu stricto. CONCLUSION: E. granulosus sensu stricto is the prevailing species of E. granulosus sensu lato in the region and pointed out the importance of sheep/dog cycle in human transmission.

Effect of oxidative stress on vital indicators of Acanthamoeba castellanii (T4 genotype).

Acanthamoeba has 22 genotypes with the T4 genotype being the main causative agent of amoebic granulomatous encephalitis and keratitis. Because the molecular mechanisms of the immune defenses of neutrophils and macrophages against histoparasites are based on oxidative stress, parasites may rely on their antioxidant systems to preclude immune defenses. Therefore, understanding of the effect of oxidative stress on vital characteristics of Acanthamoeba castellanii (T4 genotype) and the antioxidant defense responses of Acanthamoeba to oxidative status will cast light on immune cell-parasite interactions. Acanthamoeba T4 cells were cultured in RPMI-1640 medium containing different concentrations of hydrogen peroxide (H2O2). The survival of Acanthamoeba was evaluated by MTT assay and the IC50 concentration was calculated. The total antioxidant capacity (TAC) of the parasite was determined by the cupric reducing antioxidant capacity (CUPRAC) method. Malondialdehyde (MDA) as a marker of lipid peroxidation, protein carbonyl content as a measure of oxidized protein, total thiol (-SH) groups present on proteins as a major source of cellular antioxidants, and total oxidant status (TOS) were evaluated by colorimetric methods. The reactive oxygen species level increased markedly after induction of oxidative stress by the treatment of Acanthamoeba T4 with H2O2. Exposure to H2O2 also significantly increased the MDA and protein carbonyl content. The TOS level and total thiol groups also increased in the treated group compared to those in untreated parasites, although the results were not statistically significant. The TAC level was found to be significantly higher in H2O2-treated parasites, confirming that the parasite fosters its total antioxidant capacity to overcome oxidative conditions. This study showed that under oxidative stress, the defense reactions of the parasite are in part mediated by increasing its antioxidant activity, which is important for the survival of the parasite.

Ectoparasites of Rodents Captured in Hamedan, Western Iran.

BACKGROUND: Rodents with a population greater than the entire population of other mammals on earth are the source of economic losses and health conflicts. One of the major health problems with the rodents is their role as reservoir hosts of zoonotic diseases. The aim of this study was to assess the infestation of commensal rodents with ectoparasites in Hamedan City, Western Iran. METHODS: The samples were collected by live traps during years 2012-2013. After transferring the samples to the Entomological Laboratory of Hamedan University of Medical Sciences, their ectoparasites were collected and identified. RESULTS: A total of 171 slides were prepared from 105 captured commensal rodents: Mus musculus, Rattus rattus and R. norvegicus comprising three orders namely Mesostigmata: Hypoaspis (Laelaspis) astronomica, Dermanyssius sp, Pachylaelapidae (male). Metastigmata: Rhipicephalus sp and Anoplura: Polyplax spinulosa were recovered in Hamedan City. Seventy (66.6%) rodents were found infested with at least one species of ectoparasites. CONCLUSION: The results of our study indicate that ectoparasites infestation in commensal rodents of Hamedan city is high and more attention by local health authorities is needed to prevent zoonotic diseases.

Disseminated, fatal Trichosporon asahii infection in a bone marrow transplant recipient.

Trichosporon asahii is the most important species regularly isolated from systemic mycoses and shows a predilection for hematogenous dissemination. This report describes the first fatal case of disseminated trichosporonosis caused by T. asahii in a patient with familial aplastic anemia (AA). An 11-year-old girl with familial AA received chemoradiotherapy and immunosuppressive therapy for bone marrow transplantation. She was neutropenic and suffered from fever, cough, and severe mouth ulcers. T. asahii was repeatedly demonstrated by appropriate morphological and physiological characteristics, i.e., arthroconidium formation, urease activity, and assimilation of carbon and nitrogen compounds. T. asahii was found in samples of sputum, nose, and mouth ulcers by direct microscopy and culturing. Furthermore, postmortem histopathology study revealed vast tissue invasion of fungal hyphae characteristic of Trichosporon in the lung and liver. Disseminated trichosporonosis should be suspected in immunocompromised patients when a febrile condition does not improve after prolonged treatment with broad-spectrum parenteral antibiotics.

Balamuthia mandrillaris stimulates interleukin-6 release in primary human brain microvascular endothelial cells via a phosphatidylinositol 3-kinase-dependent pathway.

Balamuthia mandrillaris is an emerging protozoan parasite that can cause fatal granulomatous encephalitis. Haematogenous spread is a likely route prior to entry into the central nervous system (CNS), but it is not clear how circulating amoebae cross the blood-brain barrier. Using human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier, we determined HBMEC inflammatory response to B. mandrillaris and the underlying mechanisms associated with this response. We demonstrated that HBMEC incubated with B. mandrillaris released significantly higher levels of interleukin-6 (IL-6) (>400 pg/ml) as compared with less than 50 pg/ml in HBMEC incubated alone. Western blotting assays determined that B. mandrillaris specifically activates phosphatidylinositol 3-kinase (PI3K). By using LY294002, a PI3K inhibitor, as well as by using HBMEC expressing dominant-negative PI3K, we have identified PI3K as an important mediator of B. mandrillaris-mediated IL-6 release. We conclude that B. mandrillaris induces HBMEC signalling pathways, which lead to IL-6 release. This is the first time PI3K has been shown to play a crucial role in B. mandrillaris-mediated IL-6 release in HBMEC.

Acanthamoeba isolates belonging to T1, T2, T3, T4 but not T7 encyst in response to increased osmolarity and cysts do not bind to human corneal epithelial cells.

Acanthamoeba is an opportunistic protozoan that is widely distributed in the environment and can cause human infections. The life cycle of Acanthamoeba consists of an infective trophozoite form. However under harsh environmental conditions trophozoites differentiate into a double-walled, metabolically inactive and resistant cyst form. Research in Acanthamoeba has mostly focussed on the infective trophozoite form and its pathogenic mechanisms. In this study, we used Acanthamoeba isolates belonging to T1, T2, T3, T4, T7 genotypes and studied their cysts properties. We determined that food deprivation stimulates encystment in Acanthamoeba isolates belonging to T1, T2, T3, T4 and T7 genotypes in a sodium dodecyl sulfate (SDS)-resistant manner. In addition, increase in osmolarity triggered encystment in T1, T2, T3, T4 isolates (SDS-resistant) but T7 failed to encyst (SDS-labile). Adhesion assays revealed that Acanthamoeba cysts belonging to T1, T2, T3, T4, and T7 genotypes exhibited no and/or minimal binding (<5%) to the host cells. Fluorescein-labelled lectins showed that all Acanthamoeba isolates tested exhibited binding to concanavalin A, indicating the expression of mannosyl- and/or glucosyl-residues. Role of cysts in the transmission of infection is discussed further.

Fumonisin production by Fusarium species isolated from freshly harvested corn in Iran.

Fifty-one strains of Fusarium verticillioides and F. proliferatum isolated from corn collected from four different geographic areas in Iran, namely Fars, Khuzestan, Kermanshah and Mazandaran (an endemic oesophageal cancer (OC) area) were evaluated for their ability to produce fumonisins B1 (FB1), B2 (FB2) and B3 (FB3) in corn culture. Fumonisin levels were determined by high-performance liquid chromatography. All tested strains of F. verticillioides and F. proliferatumproduced fumonisins within a wide range of concentrations, 197-9661 microg/g, 18-1974 microg/g, and 21-1725 microg/g for FB1, FB2, and FB3, respectively. The highest mean concentrations of FB1, FB2, and FB3 were 3897, 806 and 827 microg/g, respectively. Overall, 61% of the F. verticillioides and F. proliferatum strains produced higher levels of FB3 than FB2. The mean ratios of FB1:FB2, FB1:FB3 and FB1:total fumonisins were 8, 7 and 0.7 for F. verticillioides and 5.7, 10.7 and 0.7 for F. proliferatum, respectively. Significant differences in some of the meteorological data (rainfall, relative humidity and minimum temperature) from the four provinces were observed. Fumonisin levels produced by F. verticillioides strains isolated from Khuzestan province (tropical zone) were significantly (P < 0.01) higher than the other three provinces. This is the first report of the fumonisin-producing ability of F.verticillioides and F. proliferatum strains isolated from corn harvested from different geographic areas in Iran.

Mycoflora of Iranian maize harvested in the main production areas in 2000.

Maize is one of the most important cereals produced in, and imported into, Iran. The incidences of seed-borne fungi were determined in Iranian maize harvested in 2000 from four major production areas with different climatic conditions, namely Fars, Khuzestan, Kermanshah and Mazandaran provinces. This is the first study to compare the mycoflora of maize in the aforementioned areas. Mycological analyses showed a predominance of Fusarium species (38.5%), followed by Aspergillus species (8.7%), Rhizopus species (4.8%), Penicillium species (4.5%), Mucor species (1.1%), and four other fungal genera. Fusarium verticillioides was the most prevalent species (83% of Fusarium isolates and 52% of the total isolations), with the highest incidence in Mazandaran (59%), a region of Iran with the highest rainfall and relative humidity, high rate of esophageal cancer (EC) and high levels of fumonisins in maize. Aspergillus flavus was the most widely recovered Aspergillus species and 38% of samples were contaminated with this potentially aflatoxigenic fungus. The incidence of A. flavus was highest in Kermanshah, the province with lowest mean minimum temperature. Penicillium species were seen in all the samples and Fars had the highest incidence, with highly significant differences when compared to the other three provinces. Diplodia species were not isolated from any of the samples examined.