Arginase activity of Leishmania isolated from patients with cutaneous leishmaniasis.

Cutaneous leishmaniasis (CL) is one of the most important vector-borne parasitic diseases, highly endemic in Iran, and its prevalence is increasing all over the country. Arginase (ARG) activity in isolated Leishmania parasites from CL patients is yet to be explored. This study aimed to compare the ARG activity of isolated Leishmania promastigotes from CL patients with a standard strain of Leishmania major and its influences on the disease pathogenesis. We recruited 16 confirmed CL patients from Qom Province, in central Iran; after detection of Leishmania species using PCR-RFLP, we assessed the levels of ARG in the isolated promastigotes and determined the parasites' growth rate. Only L. major was identified from CL patients. The level of ARG activity in the isolated Leishmania promastigotes from CL patients was significantly higher than that obtained from the standard strain of L. major. No significant correlations between ARG activity and lesion size, number or duration were observed; in contrast, a significant negative correlation was seen between ARG level and Leishmania' growth rate. The obtained results suggest that increased ARG expression and activity in the isolated Leishmania promastigotes might contribute to the higher parasite infectivity and play a major role in the pathogenicity of the CL.

The amoebicidal activity of Ziziphus vulgaris extract and its fractions on pathogenic Acanthamoeba trophozoites and cysts.

Acanthamoeba genus includes pathogenic species which are causal agents of a severe sight-threatening infection of the eye known as Acanthamoeba keratitis (AK). Furthermore, the number of AK is worryingly increasing worldwide, mostly in contact lens users. Until present, there is a general failure to reach a fully effective treatment against AK which is mainly due to the amoebic double-walled cyst stage which forms a protective barrier against drugs. Therefore, drug discovery research towards AK treatment is a must. In this study, Ziziphus vulgaris, a native plant of Asian countries, was checked for its activity against Acanthamoeba. For this purpose and in order to determine the in vitro amoebicidal effects of Ziziphus vulgaris aqueous extract and its fractions (chloroformic, remaining aqueous and primary alcoholic) against Acanthmoeba trophozoites and cysts, activity and sensitivity assays were performed. Moreover, the toxic effect of the extract and its fractions was also tested on murine peritoneal macrophages using a colorimetric tetrazolium salt (MTT) test. The obtained results showed that the chloroformic fraction presented a higher anti-Acanthamoeba activity when compared to the other fractions (Trophozoites/cysts were eliminated, when incubated in a concentration of 50 mg/ml of the fraction, after 24 hours). The calculated active concentrations against Acanthamoeba of these extracts did not shown any high cytotoxicity levels.This study suggests that the Ziziphus vulgaris chloroformic fraction, may present compounds with relevance for the development of novel antiAcanthamoeba drugs.

Presence of Balamuthia mandrillaris in hot springs from Mazandaran province, northern Iran.

Balamuthia mandrillaris is an opportunistic free-living amoeba that has been reported to cause cutaneous lesions and Balamuthia amoebic encephalitis. The biology and environmental distribution of B. mandrillaris is still poorly understood and isolation of this pathogen from the environment is a rare event. Previous studies have reported that the presence of B. mandrillaris in the environment in Iran may be common. However, no clinical cases have been reported so far in this country. In the present study, a survey was conducted in order to evaluate the presence of B. mandrillaris in hot-spring samples of northern Iran. A total of 66 water samples were analysed using morphological and molecular tools. Positive samples by microscopy were confirmed by performing PCR amplification of the 16S rRNA gene of B. mandrillaris. Sequencing of the positive amplicons was also performed to confirm morphological data. Two of the 66 collected water samples were positive for B. mandrillaris after morphological and molecular identification. Interestingly, both positive hot springs had low pH values and temperatures ranging from 32 °C to 42 °C. Many locals and tourists use both hot springs due to their medicinal properties and thus contact with water bodies containing the organism increases the likelihood of infection. To the best of our knowledge, this is the first report on the isolation of B. mandrillaris from hot-spring sources related to human activity. Therefore, B. mandrillaris should be considered as a possible causative agent if cases of encephalitis are suspected following immersion in hot springs in addition to Acanthamoeba and Naegleria.

High occurrence of potentially-pathogenic free-living amoebae in tap water and recreational water sources in South-West Iran.

The present study was conducted to evaluate the presence of potentially pathogenic free living amoebae in drinking and recreational water sources in south-western Iran. From 75 collected water samples, 40 samples (53.3%) were positive for free living amoebae identified using morphological tools. Interestingly, all recreational waters in Ilam city included in the present study were positive for Acanthamoeba, Vahlkampfidae and Vermamoeba. Thirty percent of tap water samples in Ahvaz city were also positive for potentially pathogenic Free Living Amoebae. Moreover, the three genera identified in the present study have been previously reported as keratitis causative agents in Iran. The present research highlights the need to improve filtration methods for tap waters and to establish awareness in recreational water sources in Iran, in order to prevent Free Living Amoebae related infections. To the best of our knowledge, this is the first report of the isolation of potentially pathogenic free living amoebae including Acanthamoebae, Vermamoebae and Vahlkampfiids in the South-West of Iran.

High occurrence of potentially-pathogenic free-living amoebae in tap water and recreational water sources in South-West Iran

The present study was conducted to evaluate the presence of potentially pathogenic free living amoebae in drinking and recreational water sources in south-western Iran. From 75 collected water samples, 40 samples (53.3%) were positive for free living amoebae identified using morphological tools. Interestingly, all recreational waters in Ilam city included in the present study were positive for Acanthamoeba, Vahlkampfidae and Vermamoeba. Thirty percent of tap water samples in Ahvaz city were also positive for potentially pathogenic Free Living Amoebae. Moreover, the three genera identified in the present study have been previously reported as keratitis causative agents in Iran. The present research highlights the need to improve filtration methods for tap waters and to establish awareness in recreational water sources in Iran, in order to prevent Free Living Amoebae related infections. To the best of our knowledge, this is the first report of the isolation of potentially pathogenic free living amoebae including Acanthamoebae, Vermamoebae and Vahlkampfiids in the South-West of Iran.

Axenic cultivation and pathogenic assays of acanthamoeba strains using physical parameters.

BACKGROUND: The main goal of the present study was to set up an axenic cultivation of Acanthamoeba and assess the pathogenic ability of T4 genotypes from different clinical and environmental strains of Acanthamoeba using two physical assays. METHODS: Sixteen Acanthamoeba isolates including 10 environmental and 6 clinical strains were cultured axenically. Axenic cultivation was performed using Proteosepepton, yeast extract and glucose medium and TY-I-S33culture. Pathogenic survey was done using osmotolerance and thermotolerance assay. Briefly, differentosmolarity (0.5 M and 1 M) of non-nutrient agar plates were performed. One hundred fifty µl of axenic culture were collected and were inoculated in 1% agar medium. For thermotolerance assay 150 µl of amoebas from axenic culture were divided into fresh culture mediums. Cultures were incubated at 37°C and 42 °C. All plates were monitored for 24 h, 48 h and 72 h. RESULTS: Overall, 16 strains of Acanthamoeba isolates previously genotyped as T4 were cultivated axenically after several months. Thermotolerance and osmotolerance assay revealed that all of clinical strains, soil and animal feces strains were highly pathogenic isolates. Two dust and water strains did not grow at high temperature (42 °C) and osmolarity (1.5 M) and thus they were classified as weak pathogens. CONCLUSION: Most of T4 genotypes are highly pathogenic organisms. This is an important finding since Acanthamoeba belonging to T4 type is the predominate genotype in environmental and clinical samples. The presence of highly pathogenic Acanthamoeba may pose a risk within susceptible people.

Occurrence of Thermotolerant Hartmannella vermiformis and Naegleria Spp. in Hot Springs of Ardebil Province, Northwest Iran.

BACKGROUND: Geothermal waters could be suitable niches for thermophilic free living amoebae including Naegleria and Hartmannella. Ardebil Province, northwest Iran is popular for having many hot springs for recreational and health purposes activity. The present research is the first molecular based investigation regarding the presence of Naegleria and Hartmannella in the hot springs of Ardebil Province in Iran. METHODS: Overall, 30 water samples were taken from waters of thermal hot springs in Ardebil Province, Iran during 2010-2011. All collected samples were transferred to Dept. of Parasitology and Mycology, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Cultivation of concentrated water samples was performed using culture-enrichment method. Cloning of the target amoebae was obtained and morphological and molecular analysis was done using page key combined with two sets of primers, respectively. Sequence analysis and homology search was used for strains identification. RESULTS: Of 30 water samples, 8 (26.7%) were positive for thermotolerant Vahlkampfiids and Hartmannella based on morphological characteristics of vegetative form and double walled cysts. Cloning of the target amoebae were done successfully. Sequencing of the positive isolates revealed that the strains belonged to Naegleria (N. carteri and N. spp) and H. vermiformis. CONCLUSION: The result highlights a need for improved filtration and disinfection and periodic monitoring of recreational thermal waters in order to prevent disease related to free- living amoebae. This is the first comprehensive molecular study of thermophilic Naegleria and Hartmannella in hot springs of Iran.

Isolation and genotyping of acanthamoeba strains from environmental sources in ahvaz city, khuzestan province, southern iran.

BACKGROUND: Acanthamoeba spp. are free-living amoebae commonly found in the environmental sources such as water, soil, and air. This ubiquitous amoeba is the causative agent of amoebic keratitis (AK). The objective of the present study was to investigate the presence of Acanthamoeba spp. in water and soil sources in Ahvaz City, Khuzestan Province, southern Iran. METHODS: In general, 110 samples of water and soil were taken from different localities of Ahvaz including agricultural canals, rivers, and swimming pools. Filtration and cultivation were carried out on non-nutrient agar medium (NNA). Axenic cultivation was performed for all of positive isolates. PCR analysis was conducted on positive samples. Sequencing was done for 15 PCR products. Genotypes were identified by Blast search and homology analysis. RESULTS: Acanthamoeba spp. was found in 43 (71.6%) of samples of water and 13 (26%) soil samples. Genotyping of 15 samples proved that Acanthamoeba belonged to T4 (86.6%), T2 (6.6%), and T5 (6.6%) genotypes. CONCLUSION: TYI-S-33 medium could be better than PYG medium for Acanthamoeba axenic culture.

Genotyping of Acanthamoeba isolated from water in recreational areas of Tehran, Iran.

A comprehensive survey assessing the presence of Acanthamoeba was conducted on 50 samples from water sources in parks and public squares from 22 municipal districts of Tehran, Iran. The prevalence and genotypes of Acanthamoeba were determined by PCR and the PCR fragments of ribosomal RNA genes sequenced. Sixteen (32%) samples were positive for Acanthamoeba spp. Sequence analysis revealed that the positive isolates belonged to the T4 and T5 genotypes. Fourteen isolates (87.5%) were T4, and two (12.5%) were T5. Acanthamoeba may be a problematic organism for contact lens wearers and for immunocompromised individuals. In Iran, Acanthamoeba keratitis has increased in recent years, mainly due to poor hygiene in contact lens wearers. A thorough survey for the prevalence of this amoeba could have a significant role in prevention of disease. This is the first report of the T5 genotype from water in recreational areas of Tehran.

Isolation of free-living amoebae from sarein hot springs in ardebil province, iran.

BACKGROUND: Free-living amoebae (FLA) are a group of ubiquitous protozoan, which are distributed in the natural and artificial environment sources. The main aim of the current study was to identify the presence of FLA in the recreational hot springs of Sarein in Ardebil Province of Iran. METHODS: Seven recreational hot springs were selected in Sarein City and 28 water samples (four from each hot spring) were collected using 500 ml sterile plastic bottles during three month. Filtration of water samples was performed, and culture was done in non-nutrient agar medium enriched with Escherichia coli. Identification of the FLA was based on morphological criteria of cysts and trophozoites. Genotype identification of Acanthamoeba positive samples were also performed using sequencing based method. RESULTS: Overall, 12 out of 28 (42.9%) samples were positive for FLA which Acanthamoeba and Vahlkampfiid amoebae were found in one (3.6%) and 11 (39.3%) samples, respectively. Sequence analysis of the single isolate of Acanthamoeba revealed potentially pathogenic T(4) genotype corresponding to A. castellanii. CONCLUSION: Contamination of hot springs to FLA, such as Acanthamoeba T(4) genotype (A. castellanii) and Vahlkampfiid amoebae, could present a sanitary risk for high risk people, and health authorities must be aware of FLA presence.

First report of vannellidae amoebae (vannella spp.) isolated from biofilm source.

BACKGROUND: Members of the Vannellidae family are free-living amoebae (FLA) distributed mainly in water and soil sources. The present study reports the first isolation of this genus in the biofilm source from hospital environment in Tehran, Iran. METHODS: Biofilm samples were collected from hospital environment. Cultivation was performed in non-nutrient agar covered with a heat-killed Escherichia coli. Cloning of the suspected amoebae was done. PCR amplification and Homology analysis using the Basic Local Alignment Search Tool (BLASTn) was performed to search for the most similar reference sequences. RESULTS: Microscopic examination showed numerous fan-shaped amoebae and peculiar cysts different to the usual shape of typical FLA. Sequence analysis of the PCR- product revealed that the suspected amoebae are highly homologous with Vannella spp. gene (99% identity and 100% query coverage) available in the gene bank database. CONCLUSION: Although Vannella spp. is not proved to be pathogenic itself, but they are capable of harboring pathogenic intracellular organisms such as Microsporidian parasites. Thus, identification of such amoebae can be of clinical importance, as they could lead to transmission of other pathogens to human.

Enteric protozoan parasites in rural areas of bandar-abbas, southern iran: comparison of past and present situation.

BACKGROUND: The main goal was to address the prevalence of enteric protozoan parasites in rural areas of Bandar-Abbas, southern Iran and to compare the results with the only conducted study in 1978. METHODS: This descriptive study was performed from 2009 through 2010 on the 565 fecal samples. Formalin-ether concentration technique was performed and the analysis was carried out using Chi-square test in SPSS software version 13.5. Finally, the comparison of our results with the only previous study which was accomplished by Sheiban and Rezaeian in 1978 was done. RESULTS: The overall prevalence of the protozoan parasites was 48.8%. However, the prevalence of pathogen parasites was 23%. Previous research in 1978 showed 80.4% infectivity. The most protozoan parasites were Blastocystis hominis (25.53%), Giardia lamblia (17.2%) and Entamoeba coli (15.95%). Previous study in 1978 found Entamoeba coli as the most common protozoa. Our finding revealed that the rate of single infectivity was much higher compared to previous research. The most frequency of infection was in children. CONCLUSION: The remarkable decrease of protozoan parasites is mainly due to progress in health care in the villages; however more effort should be done with the goal of eradicating infectious agents.

Molecular Identification and Sequencing of Mannose Binding Protein (MBP) Gene of Acanthamoeba palestinensis.

BACKGROUND: Acanthamoeba keratitis develops by pathogenic Acanthamoeba such as A. palestinensis. Indeed this species is one of the known causative agents of amoebic keratitis in Iran. Mannose Binding Protein (MBP) is the main pathogenicity factors for developing this sight threatening disease. We aimed to characterize MBP gene in pathogenic Acanthamoeba isolates such as A. palestinensis. METHODS: This experimental research was performed in the School of Public Health, Tehran University of Medical Sciences, Tehran, Iran during 2007-2008. A. palestinensis was grown on 2% non-nutrient agar overlaid with Escherichia coli. DNA extraction was performed using phenol-chloroform method. PCR reaction and amplification were done using specific primer pairs of MBP. The amplified fragment were purified and sequenced. Finally, the obtained fragment was deposited in the gene data bank. RESULTS: A 900 bp PCR-product was recovered after PCR reaction. Sequence analysis of the purified PCR product revealed a gene with 943 nucleotides. Homology analysis of the obtained sequence showed 81% similarity with the available MBP gene in the gene data bank. The fragment was deposited in the gene data bank under accession number EU678895, CONCLUSION: MBP is known as the most important factor in Acanthamoeba pathogenesis cascade. Therefore, characterization of this gene can aid in developing better therapeutic agents and even immunization of high-risk people.

Small intestinal submucosa sandwich Zilver stent-grafts for TIPS: experimental pilot study in swine.

The aim of this study was the evaluation of preliminary suitability of the piglet small intestinal submucosa (SIS) sandwich Zilver endograft for experimental transjugular intrahepatic portosystemic shunt (TIPS) creation. TIPS was created in six swine. The five surviving animals underwent the follow-up portogram and were sacrificed at four weeks after TIPS creation. Gross and histologic examination was performed in all animals. TIPS creation was successful in all animals. One animal died four days after TIPS creation. In the five surviving animals, one shunt was occluded and four shunts were stenosed on the follow-up portogram. The mean percentage narrowing of the most advanced stenoses was 72% (range, 60%-100%). The most advanced stenosis was located in the parenchymal tract in three shunts and in the hepatic vein portion in one shunt. Gross and histologic examination showed abundant neointimal formation composed mainly of fibroblasts leading to the significant shunt stenoses. The piglet SIS sandwich Zilver stent-graft was found to offer only limited TIPS patency and should not be used for the definitive long-term TIPS study.