A survey of Free-Living Amoebae (FLAs) in Nasal Mucosa of Healthy Individuals using Culture-based Method

Free-living amoebas (FLAs) are facultative parasites, and some of them can be highly pathogenic for animals and humans. These protozoan are found in abundance in environmental resources such as freshwater and soil, and thus the possible colonization of such FLA is probable. Our previous studies showed that Acanthamoeba spp. can colonize nasal mucosa of immunosuppressed patients. However, there was no report of colonization of free-living amoebae in healthy individuals in Iran.  The present study was conducted to investigate the possibility that FLAs are the normal flora of the nasal mucosa. Overall, 46 nasal swab samples were collected from healthy individuals and cultured on a 1.5% non-nutrient agar (NNA) plate enriched with a lawn of heat-killed Escherichia coli. The plates were incubated at room temperature. After one week, the plates were microscopically examined, and this was continued for one month. None of the samples were found positive for FLAs. Our findings may indicate that FLAs are not the normal flora of the nasal mucosa, or their number is so limited in the nasal mucosa that they cannot be isolated by the method used. However, supplementary and comprehensive studies are recommended

Reporting of T4 Genotype of Acanthamoeba Isolates in Recreational Water Sources of Gilan Province, Northern Iran

Background: Acanthamoeba spp. is the causative agent of blindness keratitis and fatal encephalaitis. Presence of Acanthamoeba spp. in a wide variety of niches such as different water types can lead to exposure of high risk people such as contact lens wearers. The main aim of the present study was to explore the occurrence of Acanthamoeba genotypes in the recreational water sources using both morphological and molecular approaches in Gilan province, Iran.Materials and Methods: Overall, 50 samples were collected from recreational water sources including man- made and natural waters in Gilan province. Filtration and cultivation of samples was performed using non-nutrient agar. Cloning of Acanthamoeba spp. was done to eliminate bacterial and fungi contamination. PCR amplification and sequencing were performed using genus-specific primer pair. Genotype identification was based on homology analysis of 18S rRNA gene (DF3) of the obtained sequences with the available genes in the gene bank data base.Results: Out of 50 water samples, 15 (30%) were positive for Acanthamoeba trophozoites and cysts according to morphological criteria. Cloning of 13 isolates (26%) was done successfully. Molecular analysis of 13 Acanthamoeba strain revealed that all isolates were belonged to potentially pathogenic T4 genotype.Conclusion: T4 genotype is the main cause of Acanthamoeba-related infections. Presence of Acanthamoeba belonged to T4 genotype in recreational water sources is of concern for high risk people. Alarming sign and education to high risk people is of utmost importance to prevent such infections

An Experimental Model of Primary Amoebic Meningoence phalitis Due to Naegleria australiensis in Iran

Background: The main aim of the present research was to develop the experimental meningo encephalitis due to Naegleria australiensis isolated from geothermal water sources in mice model, November 2017 in Iran. Methods: Naegleria australiensis was isolated from geothermal water sources in northern Iran. The number of amoebae was adjusted to be 1×104/ml amoebae. The experimental infection was done using 3 wk old male (BALB/c) mice. Seven animals were used for experimental amebic infection and one animal was selected for the control. Intranasal (IN) and intracerebral (IC) inoculation of amoebae were done. The mice were then monitored on daily observation and as soon as they present any brain involvement they sacrificed. The brain of all animals was then dislocated and passaged in non-nutrient agar. Results: One mouse out of seven infected mice were showed clinical symptoms of meningoencephalitis. Within few hours of culture of the brain, many vegetative forms of amoebae were detected in plate culture. The other infected animals and control mice showed no clinical symptoms until day 14. After 14 d all the animals sacrificed. The culture was negative up to one month. Conclusion: The lack of brain involvement of other animals in the present study could be due to animal immune system or it may be possible that the amoebae did not reach to olfactory bulb of nostrils

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

Distribution of Genotypes Isolated from Recreational and Therapeutic Geothermal Water Sources in Southwestern Iran

A comprehensive survey was conducted along 10 km of geothermal rivers in southwestern Iran. A total of 40 water samples were tested for the presence of Acanthamoeba spp., and genotypes were determined by targeting the diagnostic fragment 3 region of the 18S rRNA gene. The pathogenic potential of all positive isolates was also identified using tolerance ability test. High occurrences of Acanthamoeba (50%) were detected in the sampling areas. Based on sequencing analysis, isolates belonging to T4 (93.7%) and T2 (6.25%) genotypes were reported. Thermo- and osmotolerance tests revealed that five strains are highly pathogenic. Since every collection site of this study was associated with high human activity, posting of warning signs, monitoring of recreational water sources, and awareness of high-risk people are of utmost importance. To the best of our knowledge, the present research is the first to report T2 genotype from geothermal water sources in Iran

Isolation of N. philippinensis and N. americana strains from irrigation waters of farmland soils in Iran

Free-living amoebae (FLA) including Acanthamoeba spp., Naegleria fowleri, and Balamuthia mandrillaris are among the many waterborne parasites. There is not much known about the possible FLA contamination of the irrigation resources watering crops and agricultural lands in this country. Forty-five water samples were collected from water canals used for irrigation of farmlands and tested for the presence of pathogenic FLA using morphological and molecular-based methodology. The ITS region of Naegleria species was sequenced and a phylogenetic tree was generated to confirm the genetic associations and taxonomic status of the identified Naegleria spp. A total of seven (15.5%) samples were positive for FLA. Molecular analyses identified two strains as N. philippinensis (isolates: PW1 and PW2) isolated from irrigation treated water. One strain showed high homology with N. americana (isolate: PW3). To the best of our knowledge, the present study is the first study to isolate the two strains of N. philippinensis and N. americana from agricultural waters in Iran. The presence of the FLA in irrigation waters should be considered a potential health threat for exposed farmers and other people

Microscopic isolation and characterization of free living amoebae (FLA) from surface water sources.in Birjand, the capital city of the South Khorasan

Background and Aim: Free living amoebae (FLA) are amphizoic protozoa that are widely found in various environmental sources. They are known to cause serious infections in human and animal nervous systems. . The aim of the current study was to determine the presence of Acanthamoeba spp in surface water sources in Birjand city employing microscopic culture analysis based on morphology features of the amoebae. Materials and Methods: In this cross-sectional study, 50 samples of surface water sources in Birjand city. including . parks pools, public squares, waterfronts, and water stations were collected and .transferred to the laboratory and were passed through nitrocellulose filter paper. The remained elements in the filters were cultured on non-nutrient agar (NNA) with 100μl Escherichia coli suspension. After a few weeks of using morphological features,the amoeba grown were identified. Results: Out of the total of 50 samples cultured on non-nutrient agar (NNA), 19 (38%) samples.were morphologically polluted with Acanthamoeba spp,. In 2 samples (4%) a colony of Vahlkampfiidae were observed. Conclusion: The results indicated that a significant percentage of surface water sources in Birjand city was contaminated with Acanthamoeba spp. It is necessary for physicians, therefore, to take into account the diseases caused by these infectious agents. Besides, local regional health professionals should take into consideration the potential role of surface stagnant water sources in transferring these infectious agents. Placing warning signs in areas contaminated with these infectious agents seems a useful measure

Morphological and Molecular Survey of Naegleria spp. in Water Bodies Used for Recreational Purposes in Rasht city, Northern Iran

Background: Naegleria spp. is a free-living amoeba of which some species includ­ing N. fowleri and N. australeinsis are highly pathogenic in human and ani­mals. These widespread amoebae could be found in different environmental sources particularly in aquatic resources of tropical and subtropical regions. The most important source of infection is via recreational water contact. Due to the lack of thorough research regarding species of Naegleria spp. in aquatic sources, the present study was conducted. Methods: In the present study, 60 samples were collected from recreational wa­ter resources of Rasht city, Guilan province, north of Iran. After filtering and culturing the samples, plates were examined by microscopic method and accord­ing to the page criteria. DNA of vahlkampfiid-positive samples were then ex­tracted using phe­nol-chlorophorm method. Amoebae genus was identified by targeting the ITS-region and sequencing based-approaches. Results: Nine (15%) samples out of a 60 total samples were positive for Naegleria spp. of which seven belonged to potentially pathogenic N. australiensis. Two other strains were belonged to non-pathogenic N. pagei. Conclusion: The present research was the first report of occurrence of N. aus­traliensis and N. pagei in Rasht city, north Iran. This study reflects the occurrence of Naegleria spp. in water sources of Guilan Province, Iran