Background & Objectives: Cold Plasma is an emerging non-thermal, chemical-free, environmentally friendly disinfection technology. Plasma-activated water has received considerable attention from researchers in recent years. Despite extensive studies on the antibacterial effects of plasma-activated water, its anti-eukaryotic effects have not been identified. In humans, Acanthamoeba causes granulomatous encephalitis, skin ulcers, and Acanthamoeba keratitis. Considering the health importance of Acanthamoeba, this study investigated the anti-amoeba effect of plasma-activated water on trophozoites and cysts of Acanthamoeba castellanii. Methods: In this study, plasma-activated water prepared by the cold atmospheric plasma method. Physicochemical properties of produced water were evaluated by measuring pH, hydrogen peroxide, nitrite, and nitrate. To assess the effect of plasma-activated water on A. castellanii, trophozoites and cysts were exposed to plasma-activated water for 0. 5, 1, 2, 3, 4, and 5 hours. Three replicates were examined each time. At the mentioned times, cell viability was calculated by trypan-blue staining and counting on a hemocytometer, and the results were statistically analyzed. Results: Based on the physicochemical results, the mean pH of plasma-activated water in this study was about 3. 4, and the amount of hydrogen peroxide, nitrate, and nitrite were 102, 737, and 36. 94 μ, M, respectively. The present study showed that plasma-activated water killed A. castellanii trophozoites after three hours of exposure and A. castellanii cysts after four hours of exposure. On the other hand, some trophozoites gradually became cysts after exposure to plasma-activated water. These cysts became more resistant to plasma-activated water and inactivated after five hours of exposure. Conclusion: In this study, for the first time, the effect of plasma-activated water on A. castellanii was investigated. The results of the present study showed that plasma-activated water is able to inactivate A. castellanii trophozoites and cysts. Therefore, plasma-activated water can be used to disinfect and inactivate A. castellanii.

AbstractIntroduction: Chlorine is an effective disinfection agent to kill pathogenic microorganisms in the municipal water treatment system. However, despite treatment, studies have shown that Acanthamoeba is isolated from different water sources in Iran. In this study, the effect of standard concentrations of chlorine used in urban water treatment systems was evaluated on the survival of Acanthamoeba castellanii and its ultrastructure. Materials and methods: Acanthamoeba trophozoites and cysts were exposed to different concentrations (1-10 ppm) of calcium hypochlorite at different times (30 minutes, 1 and 2 hours). Field emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM) were used to study the ultrastructural changes of amoebic trophozoite.Results: This study showed that conventional chlorine concentrations could not completely eliminate A. castellanii trophozoites and cysts. Cysts were more resistant to different chlorine concentrations and compared to trophozoites, fewer cysts were killed at the same chlorine concentration and exposure time. Alteration of the cell membrane permeability, decrease in the number of pseudopodia, increase in mitochondria, vacuolation of the cytoplasm, and changes in the endoplasmic reticulum were the main ultrastructural changes in the chlorine-treated amoeba.Conclusion: This study showed that standard chlorine concentrations used as a disinfectant could not eliminate the trophozoites and cysts of A. castellanii. Due to the pathogenicity of the amoeba and its role as the reservoir and transmission of microbial agents, revising the guidelines for using disinfectants such as chlorine in the treatment of urban water systems is highlighted by this study.

Background and purpose: Acanthamoeba species are ubiquitous amphizoic organisms whichcan cause lethal diseases, such as keratitis and encephalitis in domestic animals and humans. The firststage in studies related to Acanthamoeba is achieving abundant amount of amoebae in culture medium. The aim of this study was to evaluate TYM medium as a rich medium for the diagnosis of Acanthamoebakeratitis in corneal scrapes. Materials and Methods: In this experimental study, we used one species of Acanthamoeba thatwas previously genotyped. Acanthamoeba was cultured in five plates of non-nutrient agar (NNA)medium with E. coli without TYM and in five plates of non-nutrient agar containing E. coli and TYM. Amoebae growth was observed for 24 to 48 hours by invert microscope. Results: According to current results using TYM medium increased the growth of trophozoites8. 8 folds and amoebas can be isolated form medium after 24 to 48 hours. Conclusion: Due to the significant growth of Acanthamoeba in NNA improved medium, it isrecommended for agglutination and early detection of Acanthamoeba in clinical and environmentalsamples.

Background: Acanthamoeba is an opportunistic protozoan pathogen that is known to infect the cornea to produce eye keratitis and the central nervous system to produce lethal granulomatous encephalitis. The overall aim of the present study was to determine the anti-amoebic potential of natural compound Peganum harmala against the trophozoites and cysts of Acanthamoeba in vitro.Materials and Methods: In this experimental study, a clinical isolate of Acanthamoeba was cultured and genotyped. The ethanolic extract of Peganum harmala was prepared. The trophozoites and cysts were collected by washing in page's saline. Various concentrations (1.25, 2.5, 5, and 10 mg/ml) of the ethanolic extract and polyhexanide 0.02% drop as positive control were tested at three different times (24, 48 and 72 h) on trophozoites and cysts of Acanthamoebain vitro. The viability of trophozoites or cysts was tested by eozin method, MTT, and flowcytometry analysis.Results: The results revealed that alcoholic extract had remarkable inhibitory effect on the proliferation of Acanthamoeba cysts as compared to non-treated control, and the inhibition was time and dose dependent. In the presence of 10 mg/ml ethanolic extract in medium culture after 72 h, no viable trophozoites were determined and 21.10% cysts of Acanthamoeba were viable. Percentage of trophozoites and cysts viability after adding polyhexanide 0.02% drop in medium culture after 72 hours was 0% and 23.71%, respectively.Conclusion: Ethanolic extracts of Peganum harmala could be considered a new natural compound against the Acanthamoeba trophozoites and cysts. Further works are required to evaluate the exact effect of this extract on Acanthamoeba agents in animal models.

Acanthamoeba, a widely distributed free-living amoeba with 20 genotypes identified through rRNA gene sequencing, exhibits varying degrees of pathogenicity influenced by its genotype. This study focuses on assessing the prevalence of Acanthamoeba species in the surface waters of Ilam, located in western Iran, utilizing morphological analysis and sequencing of the 18S rRNA gene through the PCR method. A total of 50 water samples were collected from various regions within Ilam city, situated in the southwest of Iran. To isolate Acanthamoeba parasites from the samples, a culture method was used, and all utilized culture media were scrutinized through microscopic and molecular techniques. The parasite’s genotype was determined by sequencing a 500-bp fragment of the 18S rRNA gene. Using microscopic and molecular methods, 19 and 16 water samples tested positive, respectively. The 18S rRNA sequences revealed that the isolates belonged to the T4, T2, and T11 genotypes. This study emphasizes the presence and inclination for close contact with highly pathogenic genotypes of Acanthamoeba in the surface waters of Ilam City.

Background and Objectives: Many plant extracts have antiparasitic properties and even some of them are effective for in vivo treatment. One of the medicinal plants is Peganum harmala, which can grow from 30 to 100 cm. The aim of the present study was to determine the effect of aqueous extract of Peganum harmala on trophozoites and cysts of Acanthamoeba in vitro. Methods: In this experimental study, a sample from a patient with amoebic keratitis, was cultured with Escherichia coli bacterium in nonnutrient agar medium (NNA) 1. 5%. Then, the aqueous extract of Peganum harmala was prepared. The extract was affected on trophozoites and cysts of Acanthamoeba at three times (24, 48, and 72h) in various concentrations (1. 25, 2. 5, 5, and 10mg/ml). The percentage of live parasites, was evaluated using homocytometer lamel, MTT assay [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide], and flow cytometry method. The data were analyzed using one-way ANOVA, one sample Kolmogorov-Smirnov test, and repeated measures test The significance level was considered to be p<0. 05. Results: The extract showed dose-and time-dependent activity on trophozoites and cysts. In the presence of 10 mg/ml aqueous extract in culture medium, 32. 47% of the trophozoites and 50. 15% of the cysts, were alive after 72 hours. Conclusion: Our study indicated that the aqueous extract of the Peganum harmala, has anti-Acanthamoeba activity. Therefore, it is recommended that the effect of Peganum harmala extract be studied in animal models.

2Background and purpose: Lophomoniasis is a relatively common emerging parasitic disease caused by a pathogenic protozoan called Lophomonas, which mostly affects the lower respiratory tract (lungs and bronchi) of humans. The parasite lives symbiotically in the digestive system of insects such as cockroaches and mites. Acanthamoeba is a free-living amoeba that has two forms of trophozoite and cyst in its life cycle. The respiratory system and airways serve as passage and deployment sites for a wide range of non-pathogenic and pathogenic microorganisms, making it a suitable place for the entry and spread of different species of Acanthamoeba amoeba and the Lophomonas pathogen in the respiratory system, which can cause clinical manifestations similar to tuberculosis. Therefore, the present study aimed to investigate the frequency of these two parasites in sputum clinical samples from suspected tuberculosis patients referred to the tuberculosis laboratory in Babolsar City to rule out the two mentioned infections. Materials and methods: This descriptive-cross-sectional study was conducted on 201 sputum samples of people suspected of tuberculosis who were referred to the tuberculosis laboratory of Babolsar Health Center in Mazandaran province in 202-2023, and all the demographic and epidemiological information of the patients was recorded. In the morphological method, Giemsa staining was used for Lophomonas and Ziehl-Neelsen staining for Acanthamoeba. To identify the presence of Acanthamoeba, sputum samples were cultured in a 1.5% non-nutrient agar culture medium for 72 to 96 hours. Approximately 50 microliters of patients' sputum were added to the NNA medium, along with 20 microliters of trypticase-yeast extract and maltose (TYM) culture medium, and 20 microliters of an autoclaved Escherichia coli bacteria mixture to enrich the culture medium. The cultured plates were then placed in an incubator at 26ºC and checked daily under a light microscope for trophozoite growth. To accurately confirm the presence of parasites, DNA extraction was performed on the patient's sputum samples using the phenol-chloroform-isoamyl alcohol method. Subsequently, conventional polymerase chain reaction (PCR) was conducted using specific primers on the extracted DNA samples. Results: In the study of 201 people suspected of tuberculosis, it was found that 80 individuals (39.8%) lived in urban areas and 121 individuals (60.2%) lived in rural areas. The age range of participants varied from 7 to 88 years old. There were 113 females (56.2%) and 88 males (43.8%) in the study, with 12 individuals from Afghanistan living in urban areas. Out of the 201 sputum samples examined, a total of 23 samples (11.4%) tested positive for the Lophomonas parasite using the PCR method. Among these positive cases, 7 individuals (30.4%) resided in urban areas (Babolsar), while 16 individuals (69.5%) lived in rural areas. However, none of the samples tested positive for Acanthamoeba in this study using staining and PCR methods. Conclusion: The present study showed that due to the similarity of the clinical manifestations of tuberculosis and lophomoniasis, there is a possibility of mistaking these two diseases clinically. Therefore, it is recommended to check fresh sputum samples of people suspected of tuberculosis, especially in endemic areas, in terms of pulmonary lophomoniasis.

Objective: Free-living amoebae, including Acanthamoeba constitute a large group of amoebae that live in fresh water, salty and bitter, moist soil, carious plants, and some on the stool. They are medically important agents. It is a medically important agent. The aim of the present study is to determine the effects of aqueous and alcoholic extracts of Artemisia annuaon trophozoites and cysts of Acanthamoeba in vitro.Methods: In this experimental research, after genotyping the clinical isolate, we prepared the aqueous and alcoholic extracts of Artemisia annua. Various concentrations (1.25, 2.5, 5, and 10 mg/ml) of the aqueous and alcoholic extracts of this plant as well as artemisinin were tested at three different times (24, 48 and 72 h) on trophozoites and cysts of Acanthamoebain vitro. We evaluated viability of the parasite by trypan blue, MTT, and flow cytometry analyses.Results: We observed the anti-acanthamoeba activity of different concentrations of the Artemisia extract. In the presence of 10 mg/ml alcoholic extract in medium culture after 72 h, we observed that 30.51% trophozoites and 91.40% cysts of Acanthamoeba were viable. However, in the presence of 10 mg/ml aqueous extract of Artemisia annua, only 58.25% trophozoites and 81.53% cysts were alive in the in medium culture after 72 h.Conclusion: Aqueous and alcoholic extracts of Artemisia annua had dose- and time dependent anti-acanthamoeba activity.

In a survey from1999 to 2001, a total of 354 samples of soil and water were colleted from different areas and examined for the presence of Acanthamoeba and Naegleria spp.After sieving, filtration and centrifugation, samples were examined for the free living protozoa ( amphizoic amoeba ). Concurrently, a sediment of each sample was cultured in the non-nutrient agar medium enriched by E.coli. In the end, 10 Acanthomoeba spp. and 3 Naegleria sp. were isolated. Besides, we diagnosed one case of human Acanthomoeba infection in a person residing in the area.