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Acanthamoeba species are among the most common free-living amoeba and ubiquitous protozoa, mainly distributed in water and soil, and cause Acanthamoeba keratitis (AK) and severe visual impairment in patients. Although several studies have reported genomic characteristics of Acanthamoeba , limited sample sizes and sources have resulted in an incomplete understanding of the genetic diversity of Acanthamoeba from different sources. While endosymbionts exert a significant influence on the phenotypes of Acanthamoeba , including pathogenicity, virulence, and drug resistance, the species diversity and functional characterization remain largely unexplored. Herein, our study sequenced and analyzed the whole genomes of 19 Acanthamoeba pathogenic strains that cause AK, and by integrating publicly available genomes, we sampled 29 Acanthamoeba strains from ocular, environmental, and other sources. Combined pan-genomic and comparative functional analyses revealed genetic differences and evolutionary relationships among the different sources of Acanthamoeba , as well as classification into multiple functional groups, with ocular isolates in particular showing significant differences that may account for differences in pathogenicity. Phylogenetic and rhizome gene mosaic analyses of ocular Acanthamoeba strains suggested that genomic exchanges between Acanthamoeba and endosymbionts, particularly potential antimicrobial resistance genes trafficking including the adeF , amrA , and amrB genes exchange events, potentially contribute to Acanthamoeba drug resistance. In conclusion, this study elucidated the adaptation of Acanthamoeba to different ecological niches and the influence of gene exchange on the evolution of ocular Acanthamoeba genome, guiding the clinical diagnosis and treatment of AK and laying a theoretical groundwork for developing novel therapeutic approaches., Importance: Acanthamoeba causes a serious blinding keratopathy, Acanthamoeba keratitis, which is currently under-recognized by clinicians. In this study, we analyzed 48 strains of Acanthamoeba using a whole-genome approach, revealing differences in pathogenicity and function between strains of different origins. Horizontal transfer events of antimicrobial resistance genes can help provide guidance as potential biomarkers for the treatment of specific Acanthamoeba keratitis cases., Competing Interests: The authors declare no conflict of interest.

Acanthamoebae spp. are considered the most commonly occurring free-living amoebae (FLA) in the environment. Their high resilience enables them to thrive in different types of environments. Using purposive sampling, 80 surface water samples were collected from identified coastal sites in Mariveles, Bataan, and Lingayen Gulf (40 water samples for each). Nineteen (23.75%) of the 80 water samples yielded positive amoebic growth during the 14-day culture and microscopic examination. The polymerase chain reaction confirmed Acanthamoeba spp. DNA in isolates MB1, A3, A4, A7, C5, and D3 using JDP1 and JDP2 primer sets. Further sequencing revealed that the isolates belonged to Acanthamoeba sp., Acanthamoeba culbertsoni , Acanthamoeba castellani , and Acanthamoeba genotype T4. The sequences were deposited in GenBank and registered under accession numbers PP741651, PP767364, PP741728, PP741729, PP767365, and PP767366, respectively. Potential risk factors such as waste disposal, expansion of human settlements to coastal locations, and soil runoffs in these environments should be controlled to mitigate the proliferation of potentially pathogenic strains of FLAs., Competing Interests: The authors declare there is no conflict., (© 2024 The Authors This is an Open Access article distributed under the terms of the Creative Commons Attribution Licence (CC BY 4.0), which permits copying, adaptation and redistribution, provided the original work is properly cited (http://creativecommons.org/licenses/by/4.0/).)

Natural hot springs are ideal places and environmental matrices that offer relaxation to people and microorganisms of different types. A total of 40 surface water samples were collected from the five identified collection sites, eight water samples for each site. Collection sites are designated 200 m apart to cover the entire study site. Surface water samples were collected approximately 10-20 cm from the surface. Water samples were filtered, cultured, and microscopically observed for 14 days. After 14 days of cultivation, eight (20%) water samples revealed cystic and trophozoite stages. Polymerase chain reaction using JDP1 and JDP2 specific primers confirmed the presence of Acanthamoeba spp. from two of our isolates in the hot spring, isolates 1.1 and 5.1. Further sequencing revealed that the isolates are Acanthamoeba T20 and Acanthamoeba genotype T7. Sequences were deposited to GenBank and were assigned accession numbers PP741726 and PP741727, respectively. The isolation of Acanthamoeba spp. in hot springs has significant health implications, especially for those who use it for recreational activity. Private resort owners are highly encouraged to regularly monitor and maintain hot spring resorts to avoid future infections., Competing Interests: The authors declare there is no conflict., (© 2024 The Authors This is an Open Access article distributed under the terms of the Creative Commons Attribution Licence (CC BY 4.0), which permits copying, adaptation and redistribution, provided the original work is properly cited (http://creativecommons.org/licenses/by/4.0/).)

Free-living amoebae (FLA) are widely distributed in the environment. Among these, Acanthamoeba spp., Naegleria fowleri, Balamuthia mandrillaris, and Vermamoeba vermiformis have been reported as human pathogens with health effects ranging from lethal encephalitis to different epithelial disorders. Despite this, FLA still present many diagnostic challenges. The aim of this study was to develop a rapid and efficient multiplex real-time quantitative polymerase chain reaction (qPCR) to simultaneously detect Acanthamoeba spp., N. fowleri, B. mandrillaris, and V. vermiformis in different water sources. For the validation of the qPCR assay, 38 samples (19 tap water and 19 stagnant water sources) were analyzed. The qPCR assay accurately identified the four types of FLA with no cross-reactivity. Considering water samples with results subsequently confirmed by conventional PCR, the multiplex qPCR assay detected 18/38 (47.4%) positive samples (Acanthamoeba spp. in 44.7% and V. vermiformis in 31.6%) and growth in nonnutritive agar (NNA) cultures identified 7/38 (18.4%) positive samples. Of the tap water samples analyzed, 26.3% of samples positive for FLA were detected by growth in NNA culture whereas 31.6% were identified by qPCR. In addition, FLA were detected in 2/19 stagnant water samples (10.5%) by growth in NNA culture and in 12/19 stagnant water samples (63.2%) by qPCR. Neither N. fowleri nor B. mandrillaris was detected in the water samples analyzed. In conclusion, the qPCR developed showed its potential as a rapid tool for detection of Acanthamoeba spp., N. fowleri, B. mandrillaris, and V. vermiformis. Moreover, FLA species were detected in half of the water sources evaluated, suggesting the importance of the surveillance of these potential infectious agents.

Free-living amoebae (FLA) of the genus Acanthamoeba are ubiquitous and amphizoic protozoa that colonize aquatic and terrestrial habitats and can serve as reservoirs for other microorganisms. They are considered econoses that can cause severe and rare pathologies. Due to limited epidemiological data available, the objective of this study was to investigate the presence of Acanthamoeba in coastal wetlands of the southeast of Buenos Aires province and evaluate their association with bacteriological and environmental variables. From February 2021 to July 2022, 22 seawater samples were collected at different points along the coast of the city of Mar del Plata (Buenos Aires, Argentina). Environmental parameters were determined and physicochemical and bacteriological studies, morphological identification, cultures and molecular typification were conducted. Regardless of the environmental and bacteriological variables, the presence of Acanthamoeba spp. was molecularly confirmed in 54.54% of the samples, being the first report of these protozoa in seawater in Argentina., (Copyright © 2024 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.)

Acanthamoeba castellanii (Douglas, 1930) Page, 1967 is the type species of a widespread genus of free-living amoebae, potentially pathogenic for humans and animals. The Neff strain is one of the most widely used in biological research, serving as a model for both A. castellanii and the whole genus in general. The Neff strain, isolated in California, closely resembles another strain found in France and originally described as a separate species, Acanthamoeba terricola Pussard, 1964, but both were successively synonymized with A. castellanii. Molecular sequence analysis has largely replaced morphological diagnosis for species identification in Acanthamoeba, and rDNA phylogenies show that the Neff strain forms a distinct lineage from that of the type strain of A. castellanii. In this study, we compared the type strain of A. terricola with the Neff strain and A. castellanii, and analysed the available molecular data including new sequences obtained from A. terricola. Here we provide molecular evidence to validate the species A. terricola. The Neff strain is therefore transferred to A. terricola and should no longer be considered as belonging to A. castellanii., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Free-living amoebae (FLA) are found in diverse environments, such as soils, rivers, and seas. Hence, they can be used as bioindicators to assess the water quality based solely on their presence. In this study, we determined the presence of FLA in river water by filtering water samples collected from various sites and culturing the resulting filtrates. FLA were detected in all the water samples with varying quality grades (Grades Ι-V). The significant increase in the size of the amoebae population with the deterioration in the water quality. Monoxenic cultures of the amoebae were performed, and genomic DNAs were isolated, among which 18S rDNAs were sequenced to identify the amoeba species. Of the 12 species identified, 10 belonged to the Acanthamoeba genus; of the remaining 2 species, one was identified as Vannella croatica and the other as a species of Vermamoeba. Acanthamoeba was detected in samples with Grades Ι to VI quality, whereas the Vermamoeba species was present only in Grade Ι water. V. croatica was found exclusively in water with Grade ΙΙ quality. Following morphological observations, genomic DNA was sequenced using 16S rDNA to determine whether the species of Acanthamoeba harbored endosymbionts. Most of the isolated Acanthamoeba contained endosymbionts, among which 4 species of endogenous bacteria were identified and examined using transmission electron microscopy. This study provides evidence that the distribution of amoebae other than Acanthamoeba may be associated with water quality. However, further confirmation will be required based on accurate water quality ratings and assessments using a more diverse range of FLA.

Purpose: Acanthamoeba spp. can be found in natural and artificial environments, which reflects their high adaptability to different conditions. Based on the available data, there is scarce information about the isolation of amoeba from milk. This study aimed to investigate the probable presence of Acanthamoeba in milk used for calf feeding., Methods: 200 milk samples from 50 industrial and traditional farms were collected. The samples were filtered and cultured on the 1.5% Non-nutrient agar medium. The amoebic growth was examined with an inverted microscope daily. DNA was extracted from the positive plates, and a PCR reaction was undertaken using the primers amplifying the Acanthamoeba 18 S rRNA gene. Five samples were purified and sequenced using specific primers. Maximum likelihood reconstructions were performed using the phylogenetic program MEGA software. The osmo and thermotolerance of isolated trophozoites were examined as well., Results: Out of 200 milk samples, Acanthamoeba was isolated from 27 (13.5%). The phylogenetic tree represents that all the isolates belonged to the genotype T4. Results of thermo and osmotolerance tests showed that isolates could develop at 37 and 43 ◦C. Besides, trophozoites survived at 0.5 M mannitol and 1 M., Conclusion: For the first time, Acanthamoeba spp. were isolated from milk used to feed dairy calves. Due to Acanthamoeba's neglected role in pathogen persistence and survival, hygiene instructions should be reconsidered., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Contact lens usage has contributed to increased incidence rates of Acanthamoeba keratitis (AK), a serious corneal infection that can lead to blindness. Since symptoms associated with AK closely resemble those incurred by bacterial or fungal keratitis, developing a diagnostic method enabling rapid detection with a high degree of Acanthamoeba-specificity would be beneficial. Here, we produced a polyclonal antibody targeting the carboxylesterase (CE) superfamily protein secreted by the pathogenic Acanthamoeba and evaluated its diagnostic potential. Western blot analysis revealed that the CE antibody specifically interacts with the cell lysates and conditioned media of pathogenic Acanthamoeba, which were not observed from the cell lysates and conditioned media of human corneal epithelial (HCE) cells, Fusarium solani, Staphylococcus aureus, and Pseudomonas aeruginosa. High titers of A. castellanii-specific antibody production were confirmed sera of immunized mice via ELISA, and these antibodies were capable of detecting A. castellanii from the cell lysates and their conditioned media. The specificity of the CE antibody was further confirmed on A. castellanii trophozoites and cysts co-cultured with HCE cells, F. solani, S. aureus, and P. aeruginosa using immunocytochemistry. Additionally, the CE antibody produced in this study successfully interacted with 7 different Acanthamoeba species. Our findings demonstrate that the polyclonal CE antibody specifically detects multiple species belong to the genus Acanthamoeba, thus highlighting its potential as AK diagnostic tool., Competing Interests: The authors have declared that no competing interests exist.

Background: Acanthamoeba spp. are one of the free-living amoeba that spread worldwide causing keratitis. Owing to the increase in the use of lenses, whether for medical or cosmetic purposes, the incidence of disease increases every year. Contamination of the lenses with the Acanthamoeba trophozoites or cysts may lead to eye infection and cause sight-threatening keratitis in human. We isolated Acanthamoeba spp. from new lenses, used lenses, and contact lens disinfecting solutions and identified them based on morphological characteristics and molecular test., Methods: New and used lenses and contact lens disinfecting solutions were cultured on monogenic media. Light and scanning electron microscope was used to identify Acanthamoeba spp. morphological features. Genotype identification was also evaluated using PCR sequencing of 18S rRNA gene specific primer pair JDP1 and JDP2., Results: A hundred samples were examined, 29 (29%) were infected with Acanthamoeba spp. That belonged to two strains of Acanthamoeba (Acanthamoeba 41 and Acanthamoeba 68). 18S rRNA of the Acanthamoeba 41 had 99.69% sequence identity to Acanthamoeba castellanii clone HDU-JUMS-2, whereas Acanthamoeba 68 had 99.74% similar pattern to that of Acanthamoeba sp. isolate T4 clone ac2t4 that are morphologically identified as Acanthamoeba polyphaga. The obtained data revealed that the isolated strains belong to T4 genotype that was evolutionarily similar to strains isolated in Iran., Conclusions: Cosmetic lenses and disinfectant solutions are a major transmissible mode for infection. This genotype is common as the cause of Acanthamoeba keratitis. To avoid infection, care must be taken to clean the lenses and their preservative solutions and prevent contamination with the parasite., Competing Interests: The authors have declared that no competing interests exist.

Acanthamoeba keratitis is predominantly caused by genotype T4. We report a case of severe keratitis caused by Acanthamoeba in a 39-year-old man who had prior accidental exposure to a corrosive chemical. The patient developed central full thickness ring infiltration and epithelial defect with hypopyon that required keratoplasty. The acanthamoebae isolated from the patient exhibited thermotolerance phenotype with the capability to grow well at ambient temperature and at 42°C. Analysis of a near complete 18S rRNA gene of this isolate revealed a distinct sequence that can be unequivocally assigned to genotype T12, a rare genotype incriminated in corneal infections.

Free-living amoeba (FLA) is widely distributed in the natural environment. Since these amoebae are widely found in various waters, they pose an important public health problem. The aim of this study was to detect the presence of Acanthamoeba, B. mandrillaris, and N. fowleri in various water resources by qPCR in Izmir, Turkey. A total of (n = 27) 18.24% Acanthamoeba and (n = 4) 2.7% N. fowleri positives were detected in six different water sources using qPCR with ITS regions (ITS1) specific primers. The resulting concentrations varied in various water samples for Acanthamoeba in the range of 3.2x105-1.4x102 plasmid copies/l and for N. fowleri in the range of 8x103-11x102 plasmid copies/l. The highest concentration of Acanthamoeba and N. fowleri was found in seawater and damp samples respectively. All 27 Acanthamoeba isolates were identified in genotype level based on the 18S rRNA gene as T4 (51.85%), T5 (22.22%), T2 (14.81%) and T15 (11.11%). The four positive N. fowleri isolate was confirmed by sequencing the ITS1, ITS2 and 5.8S rRNA regions using specific primers. Four N. fowleri isolates were genotyped (three isolate as type 2 and one isolate as type 5) and detected for the first time from water sources in Turkey. Acanthamoeba and N. fowleri genotypes found in many natural environments are straightly related to human populations to have pathogenic potentials that may pose a risk to human health. Public health professionals should raise awareness on this issue, and public awareness education should be provided by the assistance of civil authorities. To the best of our knowledge, this is the first study on the quantitative detection and distribution of Acanthamoeba and N. fowleri genotypes in various water sources in Turkey., Competing Interests: The authors have declared that no competing interests exist.

Since 2003, various viruses from the subfamily Megavirinae in the family Mimiviridae have been isolated worldwide, including icosahedral mimiviruses and tailed tupanviruses. To date, the evolutionary relationship between tailed and nontailed mimiviruses has not been elucidated. Here, we present the genomic and morphological features of a newly isolated giant virus, Cotonvirus japonicus (cotonvirus), belonging to the family Mimiviridae. It contains a linear double-stranded DNA molecule of 1.47 Mb, the largest among the reported viruses in the subfamily Megavirinae , excluding tupanviruses. Among its 1,306 predicted open reading frames, 1,149 (88.0%) were homologous to those of the family Mimiviridae . Several nucleocytoplasmic large DNA virus (NCLDV) core genes, aminoacyl-tRNA synthetase genes, and the host specificity of cotonvirus were highly similar to those of Mimiviridae lineages A, B, and C; however, lineage A was slightly closer to cotonvirus than the others were. Moreover, based on its genome size, the presence of two copies of 18S rRNA-like sequences, and the period of its infection cycle, cotonvirus is the most similar to the tupanviruses among the icosahedral mimiviruses. Interestingly, the cotonvirus utilizes Golgi apparatus-like vesicles for virion factory (VF) formation. Overall, we showed that cotonvirus is a novel lineage of the subfamily Megavirinae . Our findings support the diversity of icosahedral mimiviruses and provide mechanistic insights into the replication, VF formation, and evolution of the subfamily Megavirinae . IMPORTANCE We have isolated a new virus of an independent lineage belonging to the family Mimiviridae , subfamily Megavirinae , from the fresh water of a canal in Japan, named Cotonvirus . In a proteomic tree, this new nucleocytoplasmic large DNA virus (NCLDV) is phylogenetically placed at the root of three lineages of the subfamily Megavirinae -lineages A (mimivirus), B (moumouvirus), and C (megavirus). Multiple genomic and phenotypic features of cotonvirus are more similar to those of tupanviruses than to those of the A, B, or C lineages, and other genomic features, while the host specificity of cotonvirus is more similar to those of the latter than of the former. These results suggest that cotonvirus is a unique virus that has chimeric features of existing viruses of Megavirinae and uses Golgi apparatus-like vesicles of the host cells for virion factory (VF) formation. Thus, cotonvirus can provide novel insights into the evolution of mimiviruses and the underlying mechanisms of VF formation.

Pathogenic free-living amoebae, most notably Acanthamoeba spp., are important pathogens of the human cornea. The importance of infection with free-living amoebae in cats with keratitis is currently unclear. The aim of this study was to determine the frequency of amoeba detection in corneas of cats with naturally-acquired keratitis and in the ocular surface microflora of cats without ocular disease. Clinical ophthalmic and in vivo corneal confocal microscopic examinations were performed on 60 cats with keratitis. Corneal scrapings were analyzed by amoeba culture; cytological evaluation; and Acanthamoeba, Hartmannella, and Vahlkampfia PCR assays. Following ophthalmic examination, conjunctival specimens collected from 60 cats without clinically apparent ocular disease were analyzed similarly. In one cat with ulcerative keratitis, amoeba cysts and trophozoites were detected by in vivo corneal confocal microscopy; an Acanthamoeba sp. was isolated from corneal specimens and detected by Acanthamoeba PCR assay; and suppurative corneal inflammation was present cytologically. An Acanthamoeba sp. was isolated from conjunctival specimens from one cat without clinically apparent ocular disease, but with suppurative inflammation demonstrated cytologically. Both Acanthamoeba isolates belonged to the T4 genotype. Naegleria-like amoebae were isolated in samples from two cats with keratitis and seven cats without clinical ocular disease, but amoebae were not detected by the other assays in these samples. Amoeba detection by culture was significantly (P = 0.01) associated with cytologically diagnosed corneoconjunctival inflammation. This study identified naturally-acquired Acanthamoeba keratitis in cats. Detection of Naegleria-like amoebae in samples from cats with and without keratitis is of uncertain pathological significance., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Free living amoeba of the genus Acanthamoeba are opportunist protozoan involved in corneal, systemic, and encephalic infections in humans. Most of the mechanisms underlying intraspecies variations and pathogenicity are still unknown. Recently, the release of extracellular vesicles (EVs) by Acanthamoeba was reported. However, comparative characterization of EVs from distinct strains is not available. The aim of this study was to evaluate EVs produced by Acanthamoeba from different genotypes, comparing their proteases profile and immunomodulatory properties. EVs from four environmental or clinical strains (genotypes T1, T2, T4, and T11) were obtained by ultracentrifugation, quantitated by nanoparticle tracking analysis and analyzed by scanning and transmission electron microscopy. Proteases profile was determined by zymography and functional properties of EVs (measure of nitrite and cytokine production) were determined after peritoneal macrophage stimulation. Despite their genotype, all strains released EVs and no differences in size and/or concentration were detected. EVs exhibited a predominant activity of serine proteases (pH 7.4 and 3.5), with higher intensity in T4 and T1 strains. EVs from the environmental, nonpathogenic T11 strain exhibited a more proinflammatory profile, inducing higher levels of Nitrite, tumor necrosis factor alpha and interleukin-6 via TLR4/TLR2 than those strains with pathogenic traits (T4, T1, and T2). Preincubation with EVs treated with protease inhibitors or heating drastically decreased nitrite concentration production in macrophages. Those data suggest that immunomodulatory effects of EVs may reflect their pathogenic potential depending on the Acanthamoeba strains and are dependent on protease integrity., (© 2021 International Federation for Cell Biology.)

The evolutionary history of Acanthamoeba has been substantially resolved by the 18S rDNA phylogeny which made it possible to delimit the main lines associated with some classical species. Some of them have proven to be polyphyletic, but the inappropriate use of treating under the same names unrelated strains persists. In this study, phylogenies based on the complete genes of nuclear and mitochondrial rDNA were compared, in order to verify the congruence of the different lines. Various groups can thus be identified, some of which associated with the type strains of given species. Recognizing them only by their species names would significantly reduce the current confusion, in addition to logically following basic taxonomic rules. In this manner, the well-known polyphyletic taxa A. castellanii and A. polyphaga, are restricted to the two lines specified by their type strains, while other widely used strains like Neff and Linc-AP1 that are often confused with the previous ones, can be assigned to their own lines. New species are potentially present in other groups and additional efforts are needed to delimit them.

Free-living amoebae (FLA) are widely dispersed in the environment, can cause opportunistic and non-opportunistic infections in humans and other animals. The aim of the present study was characterize FLA obtained from air-conditioners of a public hospital in the city of Florianópolis, SC, Brazil. Fifty-four dust samples were collected of air conditioners, and were inoculated on 1.5% non-nutrient agar, overlaid with layers of Escherichia coli. Subsequently the isolates were axenised in PYG growth medium. The morphological and molecular characterization of the isolates was performed, as well as the tolerance (physiological) assays were used to evaluate the pathogenic potential. The results revealed the presence of FLA in 42 (77.8%) of the collected samples. Of these, 39 (92.9%) axenic isolates of FLA were obtained for morphological and genotypic studies. All the isolates characterized belong to the genus Acanthamoeba. Nineteen (48.7%) isolates belong to the genotype T4, 16 (41.0%) to the T5 genotype and 4 (10.3%) to genotype T11. Seven (18.0%) isolates were considered potentially pathogenic in tolerance assays. These findings require attention, considering the isolation environment and immunocompromised characteristics of many hospitalized patients.

Acanthamoeba keratitis due to a genus of free-living amoebae is a severe corneal infection. Treatment of this disease is based on the combined use of antiseptics and other drugs, including azoles. We tested isavuconazole, the latest marketed azole, in vitro, against A. castellanii, A. lenticulata and A. hatchetti. Our results show that isavuconazole presents slight amoebistatic activity against A. castellanii trophozoites but no cysticidal activity. Isavuconazole could be used only in association for management of AK due to A. castellanii., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

The aim of this study was to evaluate the inflammation process that resulted from the inoculation of Wistar Rats with Acanthamoeba griffini, a virulent T3 Acanthamoeba genotype that produces keratitis. Haematoxylin and eosin, periodic acid stain, immunohistochemistry and morphometry were used to analyse tissues from rats of an Acanthamoeba keratitis (AK) model. Two weeks after inoculating the rats with A griffini trophozoites, the thickness of the stroma had diminished, followed by an increase in thickness at 4 weeks. At the latter time, an abundance of inflammatory infiltrate cells was observed, some found to express IL-1β, IL-10 and/or caspase 3. Intercellular adhesion molecule-1 was expressed in corneal blood vessels amid the abundant vascularization characteristic of the development of AK. Through an immunohistochemical technique, trophozoites were detected at 2 and 4 weeks post-inoculation. By 8 weeks, there were a low number of trophozoites and cysts and the corneas of infected rats were similar in thickness to those of the controls. Thus, the rats were capable of healing experimental AK in the present rat model. Diverse immunological mechanisms regulated the inflammatory process in acute AK induced by A griffini in a murine model., (© 2019 John Wiley & Sons Ltd.)

Purpose: Free-living amoeba (FLA) including Acanthamoeba spp., Balamuthia mandrillaris, and Naegleria are among the soil-born parasites. There are reports of FLA-related keratitis with a history of contact with soil and dust sources, particularly among the farmers. Due to lack of the previous studies on the farmland soils and a limited number of researches conducted on recreational soils in Iran, the present study was conducted., Methods: A total of 93 soil samples including farming lands and recreational places were tested for the presence of Acanthamoeba spp. Balamuthia mandrillaris, Naegleria, and Vermamoeba using morphological key and sequencing-based tools. Pathogenicity of Acanthamoeba positive strains was also evaluated. To verify genetic associations and taxonomic status of isolated amoeba, a phylogenetic tree was built by MEGA 5.05 software inferred by the 18S rRNA gene based on maximum likelihood algorithm., Results: Overall, 28 samples (30%) were contaminated with potentially pathogenic FLA, and according to the sequencing data, 17 strains were successfully sequenced. The isolated Acanthamoeba belonged to T2, T4, T5, mixed T4 and T5 contaminations, and T11. ITS sequencing revealed the occurrence of one strain of Naegleria canariensis. Four strains of Vermamoeba vermiformis were also confirmed. Morphological survey and PCR assay failed to show any positive results for Balamuthia mandrillaris. Pathogenic potential of the Acanthamoeba strains showed that T2, T4, and T11 genotypes were highly pathogenic, whereas T5 genotypes demonstrated lower pathogenic potential., Conclusion: The results indicate that soil could be a serious hazard to human health, and therefore, further studies are expected to investigate the source of infection in patients developing FLA-related diseases. The present study is the first to investigate FLA in the farmland soils in Iran and the first to report the presence of N. canariensis in the country.

Free-living amoebae of the genus Acanthamoeba have been associated with keratitis and encephalitis. Some factors related to their pathogenic potential have been described, including the release of hydrolytic enzymes, and the adhesion and phagocytosis processes. However, other factors such as their effect over the hemodynamics and microcirculation elements have not been fully investigated. This work determines the in vitro activity of potentially pathogenic environmental isolates of Acanthamoeba genotype T4 and T5 over erythrocytes and platelets. The hemolytic activity (dependent and independent of contact), as well as the production of ADP of ten environmental isolates of Acanthamoeba obtained from dental units, combined emergency showers, dust, and hospital water, were measured. Tests were carried out over erythrocytes in suspension and blood agar plates, incubated at 4 °C, room temperature and 37 °C. Erythrophagocytosis and platelet aggregation assays were also performed. Live trophozoites of all of the isolates tested showed a hemolytic activity that was temperature-dependent. Over erythrocytes in suspension, variable hemolysis percentages were obtained: a maximum of 41% and a minimum of 15%. Regarding hemolysis over agar plates, two patterns of hemolysis were observed: double and simple halos. Conditioned medium and crude extracts of trophozoites did not show hemolytic activity. Erythrophagocytosis by Acanthamoeba was also observed; however, no production of ADP was determined by the employed methodology., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Purpose: The main goal of this study was genotyping of free-living parasites and sub-grouping of pathogenic or non-pathogenic amebae obtained from Turkey's thermal springs. In so doing, distribution and abundance of possible pathogenic or causative strain for humans, which are caused by Acanthamoeba and Naegleria strains, would be elaborated. The number of extensive studies on the general occurrence and distribution of parasitic strains is very high worldwide, but there has been a paucity of information with regard to Turkey., Methods: From a total of 434 obtained thermal pool samples, free-living amebas were isolated from 148 water samples using the non-nutrient agar (NNA) culture method. Subsequently, the cultivated samples were used for DNA isolation; then 102 obtained DNA samples were subjected to PCR amplification using various primers for samples of genera Acanthamoeba and Naegleria. Ultimately, estimation of genotype or subtype was evaluated by sequencing., Results: About 29 samples that belong to Acanthamoeba and Naegleria were estimated from a total of 102 amplified PCR samples. These eukaryotic PCR products which have Acanthamoeba genus appearance, generated 26 subtypes and 3 Naegleria samples. Among the 26 Acanthamoeba genotypes, 22 aligned sequences were matched with various GenBank reference samples, while the 4 divergent genotypes were not elaborated and marked as ND. Most of the Acanthamoeba genera were determined as likely dominating groups and clustered as T form within totally eight groups. Eight, seven and three subtypes were found as T4A, T15 and T11 genotypes, respectively while the remainings were ultimately found in four groups. Results confirming the predominance of T4A, which is known the most causative form, the presence in the pools. Despite being uncommon, N. fowleri, lovaniensis and australiensis were also observed among the surveyed pools., Conclusion: The present study is descriptive and is not unique. However, this is the most comprehensive study of the molecular distribution sampling of thermophilic Acanthamoeba and Naegleria that confirmed and demonstrated their ubiquitous presence throughout Turkey. By this estimation, in some spas, the most and likely causative form Acanthamoeba including T4 and Naegleria fowleri has also been confirmed.

Acanthamoeba is a free-living amoeba that is widely distributed in the environment. It is an opportunist protist, which is known to cause rare yet fatal infection of the central nervous system (CNS), granulomatous amebic encephalitis (GAE) in humans. GAE cases are increasingly been reported among immunocompromised patients, with few cases in immunocompetent hosts. Diagnosis of GAE primarily includes neuroimaging, microscopy, cerebrospinal fluid (CSF) culture, histopathology, serology and molecular techniques. Early diagnosis is vital for proper management of infected patients. Combination therapeutic approach has been tried in various GAE cases reported worldwide. We tried to present a comprehensive review, which summarizes on the epidemiology of GAE caused by Acanthamoeba along with the associated clinical symptoms, risk factors, diagnosis and treatment of GAE among infected patients., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Acanthamoeba spp. are ubiquitous in both natural and man-made environments and have been isolated in lakes, recreational pools, tap water, and air conditioning systems. Twenty surface water (SW) samples were collected from different sampling areas of Lake Buhi. Water samples were pelleted, cultured in NNA lawned with Escherichia coli and observed microscopically. 10% of samples (2/20) were positive for amoebic growth and were furthered tested using molecular techniques. Polymerase chain reaction showed the presence of Acanthamoeba sp. DNA. The presence of potentially pathogenic Acanthamoeba sp. poses a public health concern. The formulation of policies for proper information dissemination and control measures to avert the contraction of pathogenic FLA as well as other WBPP should be seriously considered.

The representatives of genus Acanthamoeba are widespread in the environment. The presence of freeliving Acanthamoeba sp. in such mineral deposits as bentonite was shown for the first time. Identification of isolated amoeba was conducted according to morphological features of trophozoites and cysts, as well as using sequencing of gene 18S RNA (amplifier GTSA.B1). The obtained data showed that isolated amoebae belong to the genotype T4 and II morphological group (cyst size <18 μm). For its growth, ”bentonite” amoebae are intensively used bacteria of the genus Cellulosimicrobium sp. as a food substrate.

The protista Acanthamoeba is a free-living amoeba existing in various environments. A number of species among protista are recognized as human pathogens, potentially causing Acanthamoeba keratitis (AK), granulomatous amoebic encephalitis (GAE), and chronic granulomatous lesions. In this study, 10 rhizosphere samples were collected from maize and alfalfa plants in experimental station at Institute of Genetics, Microbiology and Biotechnology, Szent István University. We detected Acanthamoeba based on the quantitative real-time PCR assay and sequence analysis of the 18S rRNA gene. All studied molecular biological methods are suitable for the detection of Acanthamoeba infection in humans. The quantitative real-time PCR-based methods are more sensitive, simple, and easy to perform; moreover, these are opening avenue to detect the effect of number of parasites on human disease. Acanthamoeba species were detected in five (5/10; 50%) samples. All Acanthamoeba strains belonged to T4 genotype, the main AK-related genotype worldwide. Our result confirmed Acanthamoeba strains in rhizosphere that should be considered as a potential health risk associated with human activities in the environment.

Acanthamoeba keratitis (AK) is a rare sight-threatening corneal infection, often reporting from contact lens wearers. An asymptomatic human immunodeficiency virus (HIV)-infected Thai male without history of contact lens use complained foreign body sensation at his left eye during motorbike riding. He had neither specific keratitis symptoms nor common drugs responding, which contributed to delayed diagnosis. By corneal re-scraping, Acanthamoeba-like cysts were detected by calcofluor white staining and agar culture. The etiological agent obtained from the culture was molecularly confirmed by Acanthamoeba spp.-specific PCR, followed by DNA sequencing. The results from BLAST and phylogenetic analysis based on the DNA sequences, revealed that the pathogen was Acanthamoeba T4, the major genotype most frequently reported from clinical isolates. The infection was successfully treated with polyhexamethylene biguanide resulting in corneal scar. This appears the first reported AK case from a non-contact lens wearer with HIV infection in Thailand. Although AK is sporadic in developing countries, a role of free-living Acanthamoeba as an opportunistic pathogen should not be neglected. The report would increase awareness of AK, especially in the case presenting unspecific keratitis symptoms without clinical response to empirical antimicrobial therapy.

The burden of HIV/AIDS in Iran is not as high as in the other countries with high prevalence; however, the number of cases of HIV/AIDs is increasing in this region. According to a recent report, Iran had 5000 (1400-13,000) new cases of HIV infection with 4000 (2500-6200) AIDS-related deaths. Individuals affected by HIV/AIDS are highly susceptible for developing opportunistic infections, e.g. the cerebral complications related to pathogenic free-living amoebae and colonization of free-living amoebae (FLA) can be a serious hazard for patients living with HIV/AIDS. In the present study, a total of 70 oral and nasal mucosal samples were obtained from HIV/AIDS patients referred to the reference hospitals in Iran and tested for the presence of potentially pathogenic FLA using culture and PCR/sequencing-based methods. To discern the taxonomic status of Acanthamoeba genotypes a maximum likelihood phylogenetic tree was constructed and tolerance assays were performed for the positive Acanthamoeba strains. Among the patients with HIV/AIDS referred to the reference hospitals from 2017 to 2019, 7.1% were found positive for pathogenic free-living amoebae. Three strains (HA3, HA4, and HA5) belonged to the T4 genotype, one strain (HA1) was related to the T5 genotype assigned as A. lenticulata, and another strain (HA2) had high homology to Vermamoeba vermiformis. The tolerance assay used for Acanthamoeba strains (HA1, HA3, and HA4) classified these amoebae as highly pathogenic strains. For the most part, the encephalitis cases occurring in HIV/AIDS patients in Iran remain undiagnosed due to lack of awareness of the practitioners on the available diagnostic tools for this lethal infection; therefore, the true incidence of GAE in this region is unknown. A possible colonization with FLA should be considered in the differential diagnosis of suspected cases of CNS infections among HIV/AIDS patients. This study is the first worldwide comprehensive study attempting to isolate and identify the FLA colonization in HIV/AIDS patients. This study highlights the fact that clinicians should be aware of the differential diagnosis of cerebral disease related to FLA in patients with HIV/AIDS.

Genus Acanthamoeba is an opportunistic protozoan that is widely distributed in the environment. Within this genus, numerous species are recognized as human pathogens, potentially causing Acanthamoeba keratitis (AK). AK is a corneal disease, associated predominantly with contact lens (CL) wear; its epidemiology is related to the specific Acanthamoeba genotypes. This study reports seven CL wearer, Acanthamoeba PCR-positive patients with AK, diagnosed between January 2015 and 2018. Patients had the diagnosis of AK 1.36 months after first symptoms. Genotyping allowed the identification of six isolates of the T4 and one of the T8 genotypes. At first presentation, pseudendritiformic epithelopathy/dirty epithelium (four eyes, 57.1%), multifocal stromal infiltrates (five eyes, 71.4%), ring infiltrate (three eyes, 42.8%), and perineuritis (one eye, 14.3%) were observed. AK was healed without later recurrence in two eyes (28.5%) using triple-topical therapy, in three eyes (42.8%) following additional penetrating keratoplasty. In one patient (14.3%), AK recurred following successful application of triple-therapy and was treated successfully with repeated triple-topical therapy and in one patient (14.3%), no follow-up data were available after diagnosis. We could not observe correlation of genotype and clinical course or the necessity of corneal transplantation in our case series.

Acanthamoeba, one of free-living amoebae (FLA), remains a high risk of direct contact with this protozoan parasite which is ubiquitous in nature and man-made environment. This pathogenic FLA can cause sight-threatening amoebic keratitis (AK) and fatal granulomatous amoebic encephalitis (GAE) though these cases may not commonly be reported in our clinical settings. Acanthamoeba has been detected from different environmental sources namely; soil, water, hot-spring, swimming pool, air-conditioner, or contact lens storage cases. The identification of Acanthamoeba is based on morphological appearance and molecular techniques using PCR and DNA sequencing for clinico-epidemiological purposes. Recent treatments have long been ineffective against Acanthamoeba cyst, novel anti-Acanthamoeba agents have therefore been extensively investigated. There are efforts to utilize synthetic chemicals, lead compounds from medicinal plant extracts, and animal products to combat Acanthamoeba infection. Applied nanotechnology, an advanced technology, has shown to enhance the anti-Acanthamoeba activity in the encapsulated nanoparticles leading to new therapeutic options. This review attempts to provide an overview of the available data and studies on the occurrence of pathogenic Acanthamoeba among the Association of Southeast Asian Nations (ASEAN) members with the aim of identifying some potential contributing factors such as distribution, demographic profile of the patients, possible source of the parasite, mode of transmission and treatment. Further, this review attempts to provide future direction for prevention and control of the Acanthamoeba infection.

The genus Acanthamoeba can cause Acanthamoeba keratitis (AK) and granulomatous amoebic encephalitis (GAE). The treatment of these illnesses is hampered by the existence of a resistance stage that many times causes infection relapses. In an attempt to add new agents to our chemotherapeutic arsenal against acanthamebiasis, two Acanthamoeba isolates were treated in vitro with newly synthesized biguanide dendrimers. Trophozoite viability analysis and ultrastructural studies showed that dendrimers prevent encystment by lysing the cellular membrane of the amoeba. Moreover, one of the dendrimers showed low toxicity when tested on mammalian cell cultures, which suggest that it might be eventually used as an amoebicidal drug or as a disinfection compound in contact lens solutions.

In this work, the presence of free-living amoebae (FLA) in dishcloths collected from human activity related places was evaluated. Once in the laboratory, 6 cm 2 pieces of each dishcloth were cut and washed with Page's Amoeba Solution (PAS) in sterile tubes. After washing, the dishcloth pieces were removed, and the tubes were centrifuged (1500 rpm for 10 min). The obtained pellets were seeded onto 2% non-nutrient agar (NNA) plates, incubated at room temperature and were monitored daily an inverted microscope. Once clonal cultures were obtained (only one type of FLA observed), molecular analyses were carried out in order to characterize the isolated FLA strains at the genus/genotype level. From the 31 dishcloths which were processed, FLA strains were isolated in NNA plates in 13 the samples (13/31, 42%). However, and due to bacterial overgrowth, only six strains were characterized at the molecular level (PCR and sequencing). Among the PCR positive strains, 83.33% (5/6) of the PCR positive samples belonged to Acanthamoeba genus (80% (4/5) to genotype T4 and 20% (1/5) to genotype T11). Furthermore, one strain was identified as a member of Allovahlkampfia genus using both morphological and molecular approaches. To the best of our knowledge, this is the first report on the isolation of Allovahlkampfia genus from dishcloths and in the Spanish territory. The presence of FLA in dishcloths should raise awareness to improve hygienic strategies in food- and domestic-related environments, in order to prevent contamination with these protozoa, which are able to be pathogenic and even to act as vehicles of other pathogenic agents.

The free-living amoebae of the genus Acanthamoeba are widely investigated for their diversity and evolution. Studies usually employ biomolecular methods targeting the 18S rRNA gene, assigning strains according to a well-established genotyping system. Strains from at least four genotypes contain introns in their rDNA. By retracing the evolutionary history of these introns within the amoebae, we found that the 18S rDNA of TUMSJ-341 strain (ATCC PRA-11), assigned to the genotype T5 (A. lenticulata), proved very unusual in our analyses, not corresponding to the characteristics of the group. The sequence contains a group I intron recovered only in A. lenticulata. At BLAST, however, the intron-less 18S rDNA of TUMSJ-341 does not match with T5 strains but shows some affinity with strains from genotype T4, suggesting a new genotype. Our accurate analysis of this sequence finally revealed a mixture of variable regions, showing that such discordant results are due to the insertion into the gene of a strain T5 of a DNA fragment containing hypervariable regions specific for a T4 strain. Data presented herein indicate that this sequence is probably a chimera.

Free-living amoebae of the genus Acanthamoeba are the etiological agents of cutaneous lesions, granulomatous amoebic encephalitis (GAE) and amoebic keratitis (AK), which are chronic infections with poor prognosis if not diagnosed promptly. Currently, there is no optimal therapeutic scheme to eradicate the pathologies these protozoa cause. In this study we report the morphological and molecular identification of three species of the genus Acanthamoeba, belonging to T4 group; A. polyphaga isolated from the corneal ulcer of a patient sample of AK case; A. castellanii isolated from the contact lens of an AK patient and A. palestinensis obtained from a soil sample. The in vitro activity of chlorhexidine, itraconazole and voriconazole drugs against trophic stage was also evaluated through a colorimetric assay based on the oxidation-reduction of alamar blue. The strains in the study were sensitive to the evaluated drugs; although when determining the 50% inhibitory concentration (IC 50 ) statistically significant differences were observed. A. castellanii showed to be highly sensitive to voriconazole (0.66 ± 0.13 μM) but the least sensitive to chlorhexidine and itraconazole (8.61 ± 1.63 and 20.14 ± 4.93 μM, respectively), A. palestinensis showed the highest sensitivity to itraconazole (0.502 ± 0.11 μM) and A. polyphaga expressed moderate sensitivity to chlorhexidine and itraconazole and lower sensitivity to voriconazole (10.10 ± 2.21 μM). These results showed that species of the genus Acanthamoeba express different sensitivity to the tested drugs, which could explain the problems surrounding the establishment of a treatment of choice in the infections caused by these amoebae. We consider that although chlorhexidine and itraconazole show good activity on these amoebae and have been used in cases of AK in Mexico with acceptable results, voriconazole should be considered as the first therapeutic option of future Acanthamoeba infections that will be diagnosed in our country., (Copyright © 2019. Published by Elsevier Inc.)

Background: Acanthamoeba spp. are the causative agents of a severe keratitis occurring mainly in contact lens wearers. The genus comprises more than 24 species that are currently divided into 20 different genotypes (T1-T20) according to sequence variations in the 18S rRNA gene. The objective of this study was to identify the genotypes and sub-genotypes of Acanthamoeba isolates collected at the Parasitology Laboratory of the Public Health Institute of Chile, the only laboratory in the country where Acanthamoeba screening is performed. This is the first report of genotypic identification of clinical isolates of Acanthamoeba in Chile and one of the few in South America., Results: In this study, 114 Acanthamoeba isolates from 76 Acanthamoeba keratitis patients, obtained between 2005-2016, were genotyped. T4 was the predominant genotype; T2 and T11 genotypes, which are scarcely reported worldwide, were also identified in Chilean patients (one and two patients, respectively). This is the first report of T2 and T11 genotypes isolated from Acanthamoeba keratitis patients in South America. It is also the first report of the T2 genotype circulating in this continent. Analysis of the diagnostic fragment 3 region of the 18S rRNA gene showed 24 T4 variants, with a predominance of the sub-genotype T4/A, followed by T4/B, T4/G, T4/C and T4/D. Bayesian analysis revealed three groups among the T4 variants: two well supported groups that included 12 and 7 sub-genotypes, respectively, and a weakly supported group that included 5 sub-genotypes. Most of the predominant T4 sub-genotypes belonged to the same group, which included 71.3% of the patients, while some minority variants lied mainly in the other two clusters., Conclusions: T2, T4 and T11 genotypes were predominantly isolated from the Acanthamoeba keratitis patients in Chile. Chilean predominant T4 sub-genotypes, which have also been reported worldwide, formed a separate cluster of the minority T4 variants. This study provides useful information about the predominant genotypes and subgenotypes that would be useful in selecting suitable strains to develop immunological and/or molecular diagnostic assays in Chile.

Acanthamoeba spp. are free-living amoeba commonly found in environmental sources such as soil, water, and dust. This ubiquitous amoeba is the causative agent of amoebic keratitis (AK) and encephalitis. The present study aimed to investigate the presence of Acanthamoeba spp. in the water sources of Kermanshah city, Iran. Sixty water samples were taken from different localities of Kermanshah including agricultural canals, rivers, and swimming pools. Filtration and cultivation were carried out on non-nutrient agar medium (NNA). The axenic cultivation was performed for all of the positive isolates. PCR analysis was performed on positive samples. Sequencing was done for 12 PCR products. Genotypes were identified by blast search and homology analysis. The obtained data were analyzed using Statistical Package for the Social Sciences (SPSS 16) software. Acanthamoeba spp. was found in 46 (76.66%) water samples and amoebae were grown in the TYI-S-33 medium. Sequencing of 12 samples proved that Acanthamoeba belonged to T4 (75%), T2 (8.34%), T5 (8.33%) and T11 (8.33%) genotypes. In this study, Acanthamoeba T4 (75%), T2 (8.34%), T5 (8.33%) and T11 (8.33%) genotypes were isolated from the water of Kermanshah city. Thus, hygiene consideration is recommended to prevent the contamination.

Introduction: Free-living amoebae, ubiquitous in outer environments, in predisposing circumstances may exist as parasites, infectious agents of Acanthamoeba keratitis. In recent decades, the vision-threatening corneal infection is a growing human health threat worldwide, including Poland. The applied therapy is often ineffective due to diagnostic mistakes, various pathogenicity of Acanthamoeba strains and high resistance of cysts to drugs; many agents with possible anti-amoebic activity are still being tested. In the presented study, selected chemicals are investigated in terms of their in vitro effect on corneal and environmental Acanthamoeba strains., Material and Methods: Samples of a corneal isolate from a patient with severe Acanthamoeba keratitis,of assessed on the basis of genotype associations of 18S rRNA and the type strain, Acanthamoeba castellanii Neff cultivated in bacteria-free condition, were exposed to povidone iodine, chlorhexidine digluconate or toyocamycin. In vitro population dynamics of the strains were monitored and compared to those of control cultures., Results: All chemicals showed anti-amoebic effects with different degrees of effectiveness. Significant differences were observed in the in vitro population dynamics, and the morpho-physiological status of A. castellanii Neff T4 and corneal strains determined as A. polyphaga T4 genotype, exposed to povidone iodine or toyocamycin, in comparison with chlorhexidine taken as reference., Conclusions: Time-dependent amoebstatic in vitro effects were demonstrated for all agents, in particular, the results of assays with povidone iodine are promising. No significant stimulation of encystation appeared; however, as cysticidal efficacy of chemicals is expected, complementary research is needed on different Acanthamoeba strains with modified agent concentrations and method application.

The genus Acanthamoeba is a free-living amoeba widely distributed in various aquatic environments. It is an etiologic cause of amoebic encephalitis and keratitis particularly for immunocompromised individuals. The purpose of the present study was to investigate Acanthamoeba species prevalence in household and hospital potable water in Beni-Suef governorate, Egypt, and to employ sequencing methods to identify positive Acanthamoeba species isolates and their potential health risks. Sixty tap water samples (30 household and 30 governmental and private hospital settings) collected from Beni-Suef governorate, Egypt were filtered, cultured on non-nutrient agar, identified by morphotyping keys after staining with Giemsa stain and then confirmed by PCR using Acanthamoeba specific primers. Twenty positive samples were successfully genetically characterized and phylogenetically analyzed to identify Acanthamoeba species. The total detection rate for Acanthamoeba was 48/60 (80%); Acanthamoeba contamination in water collected from domestic houses was higher than in hospitals; 27/30 (90%) versus 21/30 (70%) with statistical significant value (P value = 0.05). Sequencing of 20 positive isolates revealed Acanthamoeba T4 in 65% and T2 in 35%. To our knowledge, this is the first research that documents the occurrence and phylogeny of Acanthamoeba species in Beni-Suef, Egypt. The presence of a higher percentage of Acanthamoeba species in tap water, in particular T4, highlights the potential health hazards for immunocompromised individuals and emphasizes the urgent need for the implementation of effective filtration and disinfection measures.

Members of genus Acanthamoeba are widely distributed in the environment. Some are pathogenic and cause keratitis and fatal granulomatous amoebic encephalitis. In this study, we isolated an Acanthamoeba CJW/W1 strain from tap water in Wuxi, Jiangsu Province, China. Its 18S rDNA was sequenced and a phylogenetic tree was constructed. The isolated cysts belonged to morphologic group II. Comparison of 18S rDNA sequences of CJW/W1 strain and other isolates showed high similarity (99.7%) to a clinical isolate Asp, KA/E28. A phylogeny analysis confirmed this isolate belonged to the pathogenic genotype T4, the most common strain associated with Acanthamoeba-related diseases. This is the first report of an Acanthamoeba strain isolated from tap water in Wuxi, China. Acanthamoeba could be a public health threat to the contact lens wearers and, therefore, its prevalence should be monitored.

Little is known about the prevalence of Balamuthia mandrillaris within the environment due to its difficult isolation, but once an axenic culture is established, it is relatively easy to maintain. As most of the time researchers are interested mainly in isolating B. mandrillaris from environmental samples, the flora that accompanies it becomes second in importance. Therefore, this study aimed to determine which potentially pathogenic free-living amoebae, in addition to B. mandrillaris, could be found co-inhabiting a source of natural thermal water called "Agua Caliente" (Mexico), where this amoeba has previously been detected twice by molecular methods. A third sampling from this same source was carried out to try to isolate B. mandrillaris and other free-living amoebae using 37 and 45 °C as isolation temperatures. For PCR techniques, specific primers were used for B. mandrillaris, Naegleria fowleri, and Acanthamoeba species, plus a universal primer set for the eukaryotic 18S SSU rRNA gene for other isolated amoebae. PCR products were sequenced for final identification. 42 strains of the primary isolate were obtained, but only 34 could be kept in culture. Of them, 23 strains were identified as Naegleria lovaniensis, eight strains as Acanthamoeba jacobsi, two strains as Stenamoeba sp. and only one was identified as Vermamoeba vermiformis. The isolation of B. mandrillaris was once again not successful, but the presence of potentially pathogenic and nonpathogenic free-living amoebae is reported for the first time in this type of water in Mexico thanks to molecular methodology., (Copyright © 2018. Published by Elsevier Inc.)

Free-living amoebae (FLA) are widely spread in the environment and also known to cause rare but often serious infections. The present work focuses on a local survey on FLA. It is essential to know the prevalence and distribution of these microorganisms in order to get infections caused by them under control. In this study, FLA isolated from domestic tap water samples from homes of contact lens wearers were identified by morphology and by 18S rRNA gene sequence analysis. Morphological analysis and partial sequencing of the 18S rDNA revealed the presence of Acanthamoeba genotype T4 and Vermamoeba vermiformis in the investigated tap water samples. Naegleria fowleri, Balamuthia mandrillaris, and Sappinia spp. were not detected during this study. It was shown that species of FLA known to cause eye infections in humans are widely distributed in tap water in Istanbul, Turkey. Contact lens wearers should be aware of the risk of contamination from tap water and strictly apply stringent contact lens hygiene. With this study, we established Acanthamoeba genotype T4 and Vermamoeba vermiformis as contaminants of tap water in Istanbul., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Various strains belonging to three Acanthamoeba species, A. griffini (genotype T3), A. lenticulata (T5), and A. jacobsi (T15), have group I introns in their 18S rRNA genes. Group I introns are self-splicing ribozymes that can spread among host lineages either through an intron-encoded endonuclease at the DNA level, or by reverse splicing during the RNA cycle. In Acanthamoeba, introns belong to the subclass IC1, they are located at one out four positions within the rRNA, show low identity values and all lack open reading frames to encode for an endonuclease. Uncharacterized introns from strains of another genotype, T4 (A. castellanii complex), resemble those of genotype T3, and at least one of them contains a non-functional endonuclease gene. Here, we analyzed all available data on Acanthamoeba 18S rDNA sequences to identify the possible presence of open reading frames that could encode endonucleases. We found a total of eight 18S rDNA sequences, all from T4 strains, that have introns containing putative non-functional endonuclease genes. Furthermore, two distinct endonucleases can be identified that are differently inserted in unrelated introns., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Acanthamoeba are free-living amoebae found in various ecological environments, and they are major microbial predators. Some Acanthamoeba species can cause keratitis, granulomatous amoebic encephalitis, and infections of the organs. This is the first study in Latvia describing Acanthamoeba detection by molecular techniques and genotyping using tap water from different sources. An 18S rRNA gene investigation of the 20 Acanthamoeba isolates suggested that 19 belonged to the hazardous T4 genotype and one sample was T12. The results indicate that tap water in Latvia may be a source of Acanthamoeba that is potentially pathogenic to humans. All obtained sequences were submitted to the GenBank database under the following accession numbers: MG386295-MG386314.

Acanthamoeba spp. is a widespread protozoan that has been isolated from air, dust, soil, water and biological samples. An opportunistic pathogen of humans and animals, it may cause ocular keratitis, encephalitis, and even multisystem disease. The frequency of Acanthamoeba in animals is unknown. The aim of present study was determine the presence of Acanthamoeba spp. in immunocompromised stray cats - animals possibly more likely to harbour the infection given their immunocompromised status and frequenting of contaminated environments. Of 307 cats examined, 55 were positive for feline immunodeficiency virus and/or feline leukaemia virus and therefore included in the study. Corneal scrapings were obtained to isolate Acanthamoeba spp. by culture and molecular detection by conventional and real time PCR. None of the samples examined directly by molecular methods were positive for Acanthamoeba spp. However, two (3.6%) cases of the cultured samples provided positive results, which were confirmed by subsequent molecular analysis. Sequencing assigned one isolate to genotype T4 and the other to T2. Since Acanthamoeba spp. may also infect animals and humans, the present findings may raise some public health and veterinary concerns., (Copyright © 2018. Published by Elsevier Inc.)

Acanthamoeba keratitis (AK) is a severe corneal infection that may occur as a serious outcome of improper use of contact lenses (CL)., Objectives: The study aimed to diagnose AK in soft CL users presenting with infectious keratitis, and to identify the prevalent genotypes isolated from different cases. Another aim was to determine the CL hygiene-related risk behaviors, and to explore the risk of water exposure for developing AK., Methods: A cross sectional study was performed. A questionnaire was carried out including 260 clinically diagnosed cases as infectious keratitis (170 females and 90 males); all of them were soft CL users for the suspected risk factors. Corneal scrapes from the affected eyes were cultured to diagnose bacterial and AK. PCR was performed and the amplified products were sequenced and compared with GenBank data., Results: The parasite was positively amplified from 32 samples (12.3%). Acanthamoeba T4 genotype was identified in 27/32 (84.4%) of isolates. Other detected genotypes belonged to T5 and T3 genotypes at rates of 9.4%, and 6.25%, respectively. The most important risk factors associated with development of AK were female sex, sleeping while wearing CL, and exposure to water resources through different practices. These practices included rinsing the CL case in tap water, swimming and/or showering while wearing CL, using multipurpose solution for cleaning the lenses, using water from over-building tanks. Rubbing the eyes due to discomfort when applying CL was an additional important risk factor associated with AK. The protective factor was regular hand washing before using CL., Conclusion: CL users were more exposed to AK and should gain enough health education regarding proper lens hygiene and dangers of tap water exposure., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Free-living amoeba of the genus Acanthamoeba can eventually act as parasites, causing infections in humans. Some physiological characteristics of Acanthamoeba have been related to the grade of pathogenicity, allowing inferences about the pathogenic potential. The main goal of this study was to characterize isolates of Acanthamoeba obtained in Brazil and evaluate properties associated with their pathogenicity. A total of 39 isolates obtained from keratitis cases (n = 16) and environmental sources (n = 23) were classified into morphological groups and genotyped by sequencing the 18S rDNA fragments ASA.S1 and GTSA.B1. Samples were also tested regarding their thermo-tolerance, osmo-tolerance, and cytopathogenicity in MDCK cells. Isolates were identified and classified as follows: group I (T17, T18); group II (T1, T3, T4, T11); and group III (T5, T15), with the predominance of genotype T4 (22/39). Clinical isolates were genotyped as T3 (1/16), T4 (14/16) and T5 (1/16). The majority of isolates (38/39) were able to grow at 37 °C, but tolerance to 40 °C was more frequent among environmental samples. The tolerance to 1 M mannitol was infrequent (4/39), with three of these corresponding to clinical samples. The variable ability to cause cytopathic effects was observed among isolates of distinct genotypes and origins. This study identified, for the first time, T1 and T18 in Brazil. It also indicated a weak association between the clinical origin of the isolates and tolerance to high temperatures, high osmolarity, and cytopathogenicity, demonstrating that some in vitro parameters do not necessarily reflect a higher propensity of Acanthamoeba to cause a disease.

In the southern Tunisia Oasis, we conducted 211 water with drawals from various water traffic sites. This water is used for agriculture, swimming or various other human activities. Acanthamoeba genus was detected in 82% of collected samples. Sequencing of the amplification products with primers P892C/P892 has allowed us to detect genotypic variation with predominance of T4 genotype (51%) and presence of the genotypes T14, T5, T3, T16, T15, T10, T11, T9 and T7. They T4, T3, T5, T15, T11 and T10 genotypes have a high potential for pathogenicity and a very high degree of virulence due to their production of serine proteases and extracellular cysteine enzymes involved in tissue degradation of the host. T4 genotype was the most abundant in the environment as well as in infections caused by Acanthamoeba spp. T5 genotype was ranked second and T3 genotype was less abundant in the environment and its pathogenicity is discussed. Acanthamoeba strains with the genotypes T16, T9 and T7 were considered non pathogenic. In fact, they have been isolated only from the environment. However, for these strains, their role as a reservoir can be a real risk to human health., (Copyright © 2018. Published by Elsevier Inc.)

Acanthamoeba spp. are ubiquitous in the environment and have the potential to cause severe infections. The different genotypes of Acanthamoeba have been shown to influence the severity of the disease and response to therapy. Characterizing Acanthamoeba spp. upto genotype can aid in infection control practices. Twenty-five Acanthamoeba isolates, characterized by 18S rDNA sequencing, were subjected to MALDI-TOF MS analysis by creating a database for the individual genotypes. The differentiating features of the various spectra were observed; the coded samples were then tested against the created database. The results of identification were compared with sequencing. Five different genotypes were obtained-T3, T4, T5, T10, and T11. Spectral analysis revealed genus-specific and genotype-specific peaks. The peak patterns for individual genotype were discrete and reproducible. Clinical isolates produced different peaks from the environmental isolate of the same genotype. A concordance of 92% was obtained with MALDI-TOF MS in comparison with 18sDNA sequencing. MALDI-TOF MS, once optimized, has the potential to reliably identify the genotype of Acanthamoeba spp. and to differentiate clinical isolate from mere contaminant.

Acanthamoeba has a worldwide distribution in the environment and it is capable of causing a painful sight-threatening disease of the cornea designated as Acanthamoeba keratitis (AK). Nowadays, the cases of AK have surged all over the world along with its disease burden due to increasing use of contact lenses used not only for optical correction but also for cosmetic purposes. In our present work, epithelial abrasion of a 27-year-old female soft contact lens wearer with keratitis was examined. Genotype identification was carried out with a real-time fluorescence resonance energy transfer polymerase chain reaction (PCR) assay based on sequence analysis of the 18S rRNA gene. Genotyping allowed the identification of a T8 group isolate. The analysis confirmed the importance of a complete diagnostic protocol, including a PCR assay, for the clinical diagnosis of AK from human samples. Acanthamoeba T8 should be considered as potential causative organism in keratitis in human.

Free-living amoebae of the genus Acanthamoeba are potentially pathogenic protozoa widespread in the environment. The detection/diagnosis as well as environmental survey strategies is mainly based on the identification of the 18S rDNA sequences of the strains that allow the recovery of various distinct genotypes/subgenotypes. The accurate recording of such data is important to better know the environmental distribution of distinct genotypes and how they may be preferentially associated with disease. Recently, a putative new acanthamoebal genotype T99 was introduced, which comprises only environmental clones apparently with some anomalous features. Here, we analyze these sequences through partial treeing and BLAST analyses and find that they are actually chimeras. Our results show that the putative T99 genotype is very likely formed by chimeric sequences including a middle fragment from acanthamoebae of genotype T13, while the 5'- and 3'-end fragments came from a nematode and a cercozoan, respectively. Molecular phylogenies of Acanthamoeba including T99 are consequently erroneous as genotype T99 does not exist in nature. Careful identification of Acanthamoeba genotypes is therefore critical for both phylogenetic and diagnostic applications.