Acanthamoeba Species Research Articles

Lethal encounters: The evolving spectrum of amoebic meningoencephalitis.

The free-living amoebae are thermophilic organisms that may play an increasing role among diseases of a warming world. They are uncommon, accidental, yet high consequence pathogens, with differing pathologic syndromes. New prospects for diagnosis and life-saving treatment make early disease recognition imperative. We review the three most commonly diagnosed species that infect humans: Naegleria fowleri, Acanthamoeba species, and Balamuthia mandrillaris.

In Vitro Evaluation of Adhesion of Two Acanthamoeba Strains to Cosmetic Contact Lenses.

To evaluate the factors affecting the adhesion of Acanthamoeba trophozoites to the surface of cosmetic contact lenses (CCLs). Acanthamoeba castellanii and A. hatchetti trophozoites were inoculated onto CCLs (hema copolymer [HM] [38.5% H2O], phemfilcon [PF] [55% H2O], polymacon [PM] [38% H2O], polyhema [PH] [%42 H2O], and hema [HM55] [55% H2O]), and the number of trophozoites adhered to the lens surfaces was assessed over time, that is, at 15 min, 1, and 24 hr. In addition, scanning electron microscopy (SEM) analysis of the lens surfaces was performed to evaluate the effect of lens surface topology on adhesion. The number of amoeba adhered to the contact lens surface was found lower with PF and PH production materials, than lenses with HM, PM, and HM55 production materials (P<0.05). No significant difference was detected in amoebic strains adhered in all the contact lens types (P>0.05). No significant difference was found on average amoeba adhesion between contact lenses with hema production material but with different water contents (45%, 55%), to see the effect of water content on amoebic adhesion (P>0.05). As a result of SEM analysis, surface topology showed no effect on adhesion. (1) Chemical composition of lenses seemed to be mostly responsible for the adhesion of Acanthamoeba. (2) Different numbers of trophozoites, obtained after the adhesion experiment, could also indicate that adherence capacity can also differ among Acanthamoeba species.

Potentially pathogenic genera of free-living amoebae coexisting in a thermal spring

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.

Comparative in vitro effectiveness of a novel contact lens multipurpose solution on Acanthamoeba castellanii

BackgroundA multipurpose contact lens cleaning solution (MPS) containing novel active ingredients under development was compared to two commercially available MPS solutions for effectiveness against Acanthamoeba isolates.MethodsThe Acanthamoeba isolate A. castellanii was propagated for trophozoite or cyst-containing cultures for the purpose of assessment of effectiveness of each MPS. An alamar blue-based cellular respiration assay was used to assess effectiveness against trophozoites; Trypan blue hemocytometer-based microscopic counts measured cysticidal effects. To assess the general antimicrobial potency of each solution as controls for the anti-amoebic assays, comparative bactericidal effectiveness using Serratia marcenses was also performed.ResultsMinimal effectiveness against either Acanthamoeba form was observed from either commercial MPS. In contrast, the novel MPS achieved complete kill within 1 h contact time for both Acanthamoeba trophozoite and cysts. Each commercial MPS required 6 h contact time to achieve a two to three log reduction in S. marcenses. In contrast, the experimental MPS achieved disinfection in 60 min contact time, and complete kill (< 1 CFU) at 90 min.ConclusionsResults suggest that the inclusion of a novel ingredient combination within the MPS under development clearly is required and is ideal for rapid and effective killing of Acanthamoeba species in the context of contact lens disinfection systems. The representative commercially available MPS used in this testing provided minimal effectiveness against the protozoa regardless of contact time. In addition, comparative results with the bacterial agent in the control study show distinct differences in the speed to disinfection with the novel MPS. Future MPS development should consider inclusion of novel chemical entities that are effective against Acanthamoeba species to speed disinfection and further reduce the exposure potential of users of contact lenses and cleaning systems.

Clinically Approved Drugs against CNS Diseases as Potential Therapeutic Agents To Target Brain-Eating Amoebae.

Central nervous system (CNS) infections caused by free-living amoebae such as Acanthamoeba species and Naegleria fowleri are rare but fatal. A major challenge in the treatment against the infections caused by these amoebae is the discovery of novel compounds that can effectively cross the blood-brain barrier to penetrate the CNS. It is logical to test clinically approved drugs against CNS diseases for their potential antiamoebic effects since they are known for effective blood-brain barrier penetration and affect eukaryotic cell targets. The antiamoebic effects of clinically available drugs for seizures targeting gamma-amino butyric acid (GABA) receptor and ion channels were tested against Acanthamoeba castellanii belonging to the T4 genotype and N. fowleri. Three such drugs, namely, diazepam (Valium), phenobarbitone (Luminal), phenytoin (Dilantin), and their silver nanoparticles (AgNPs) were evaluated against both trophozoites and cysts stage. Drugs alone and drug conjugated silver nanoparticles were tested for amoebicidal, cysticidal, and host-cell cytotoxicity assays. Nanoparticles were synthesized by sodium borohydride reduction of silver nitrate with drugs as capping agents. Drug conjugated nanoconjugates were characterized by ultraviolet-visible (UV-vis) and Fourier transform infrared (FT-IR) spectroscopies and atomic force microscopy (AFM). In vitro moebicidal assay showed potent amoebicidal effects for diazepam, phenobarbitone, and phenytoin-conjugated AgNPs as compared to drugs alone against A. castellanii and N. fowleri. Furthermore, both drugs and drug conjugated AgNPs showed compelling cysticidal effects. Drugs conjugations with silver nanoparticles enhanced their antiacanthamoebic activity. Interestingly, amoeba-mediated host-cell cytotoxicity was also significantly reduced by drugs alone as well as their nanoconjugates. Since, these drugs are being used to target CNS diseases, their evaluation against brain-eating amoebae seems feasible due to advantages such as permeability of the blood-brain barrier, established pharmacokinetics and dynamics, and United States Food and Drug Administration (FDA) approval. Given the limited availability of effective drugs against brain-eating amoebae, the clinically available drugs tested here present potential for further in vivo studies.

Molecular characterization and phylogenetic analysis of Acanthamoeba isolates in tap water of Beni-Suef, Egypt.

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.

A Phylogenomic Study of Acanthamoeba polyphaga Draft Genome Sequences Suggests Genetic Exchanges With Giant Viruses.

Acanthamoeba are ubiquitous phagocytes predominant in soil and water which can ingest many microbes. Giant viruses of amoebae are listed among the Acanthamoeba-resisting microorganisms. Their sympatric lifestyle within amoebae is suspected to promote lateral nucleotide sequence transfers. Some Acanthamoeba species have shown differences in their susceptibility to giant viruses. Until recently, only the genome of a single Acanthamoeba castellanii Neff was available. We analyzed the draft genome sequences of Acanthamoeba polyphaga through several approaches, including comparative genomics, phylogeny, and sequence networks, with the aim of detecting putative nucleotide sequence exchanges with giant viruses. We identified a putative sequence trafficking between this Acanthamoeba species and giant viruses, with 366 genes best matching with viral genes. Among viruses, Pandoraviruses provided the greatest number of best hits with 117 (32%) for A. polyphaga. Then, genes from mimiviruses, Mollivirus sibericum, marseilleviruses, and Pithovirus sibericum were best hits in 67 (18%), 35 (9%), 24 (7%), and 2 (0.5%) cases, respectively. Phylogenetic reconstructions showed in a few cases that the most parsimonious evolutionary scenarios were a transfer of gene sequences from giant viruses to A. polyphaga. Nevertheless, in most cases, phylogenies were inconclusive regarding the sense of the sequence flow. The number and nature of putative nucleotide sequence transfers between A. polyphaga, and A. castellanii ATCC 50370 on the one hand, and pandoraviruses, mimiviruses and marseilleviruses on the other hand were analyzed. The results showed a lower number of differences within the same giant viral family compared to between different giant virus families. The evolution of 10 scaffolds that were identified among the 14 Acanthamoeba sp. draft genome sequences and that harbored ≥ 3 genes best matching with viruses showed a conservation of these scaffolds and their 46 viral genes in A. polyphaga, A. castellanii ATCC 50370 and A. pearcei. In contrast, the number of conserved genes decreased for other Acanthamoeba species, and none of these 46 genes were present in three of them. Overall, this work opens up several potential avenues for future studies on the interactions between Acanthamoeba species and giant viruses.

Co-culture models illustrate the digestion of Gemmata spp. by phagocytes

Gemmata spp. bacteria thrive in the same aquatic environments as free-living amoebae. DNA-based detection of Gemmata spp. sequences in the microbiota of the human digestive tract and blood further questioned the susceptibility of Gemmata spp. to phagocytes. Here, Gemmata obscuriglobus and Gemmata massiliana were co-cultured with the amoebae Acanthamoeba polyphaga, Acanthamoeba castellanii, Acanthamoeba griffini and THP-1 macrophage-like phagocytes. All experiments were performed in five independant replicates. The ratio amoeba/bacteria was 1:20 and the ratio THP-1/bacteria was 1:10. After a 2-hour co-culture, extracellular bacteria were killed by kanamycin or amikacin and eliminated. The intracellular location of Gemmata bacteria was specified by confocal microscopy. Microscopic enumerations and culture-based enumerations of colony-forming units were performed at T = 0, 1, 2, 3, 4, 8, 16, 24, 48 and 72 hours post-infection. Then, Gemmata bacteria were engulfed into the phagocytes’ cytoplasmic vacuoles, more than (98 ± 2)% of Gemmata bacteria, compared to controls, were destroyed by phagocytic cells after a 48-h co-culture according to microscopy and culture results, and no positive culture was observed at T = 72-hours. Under our co-culture conditions, Gemmata bacteria were therefore susceptible to the environmental and host phagocytes here investigated. These data suggest that these Acanthamoeba species and THP-1 cells cannot be used to isolate G. massiliana and G. obscuriglobus under the co-culture conditions applied in this study. Although the THP-1 response can point towards potential responses that might occur in vivo, these responses should first bevalidated by in vivo studies to draw definite conclusions.

Isolation of Balamuthia mandriallaris parasite from air conditioning of the houses in south and southeast of Turkey

The free living amoebae cause various infections such as Acanthamoeba keratitis, granulomatous amoebic encephalitis, primer amoebic meningoencephalitis in humans and animals. The free living amoebae Acanthamoeba, Balamuthia mandriallis, Naegleria fowleria and Sappinia species that cause disease in humans have been isolated from many environmental materials until today. However, no isolation has been reported from the filters of the air conditions from the houses used for ventilation. The aim of this study was toinvestigate the existence of free living amoebae using molecular methods in the filters of air-conditions used in the study living area of the people. A total of 30 dust samples were taken from the filtersof air-conditions in Adana and Gaziantep province of Turkey. DNA isolation of the dust samples was performed using the DNeasy PowerSoil kit (Qiagen, Germany) and polymerase chain reaction was done with specific primers of Acanthamoeba spp., B.mandriallis, N.fowleria and Sappinia species. As a result of this study, Acanthamoeba spp. was determined as 33.3% (5/15) and B.mandriallis was determined as6.6% (1/15) in Adana province. On the other hand, Acanthamoeba species was determined as 26.6% (4/15) and B.mandriallis was determined as 13.3% (2/15) in Gaziantep province. N.fowleria and Sappina species were not detectedin both of the cities. DNA sequence analysis was performed for the confirmation of the species and 99% of the results were similar to the other species in GenBank. The rates of Acanthamoeba castellanii and Acanthamoeba griffinii (T3) were determined as %66.6 (6/9) and 33.3% (3/9), respectively by DNA sequencing. Distribution of Acanthamoeba species according to the cities were 33.3% (3/9) for A.castellanii and 22.2% (2/9) for A.griffini in Adana. It was 33.3% (3/9) for A.castellanii and 11.1% (1/9) for A.griffini in Gaziantep. There was no significant difference in the distribution of the parasite species among cities (p> 0.1). It is important to raise awareness of the diseases caused by free living amoebae among people. Acanthamoeba species have been reported frequently from environmental materials in Turkey, but B.mandriallis has not been reported from any environmental sample since this study. The presence of B.mandriallis has been reported in the air-conditions of houses in this study. This result shows that people have risk in terms of illness of free living amoebae in living areas. Our study emphasized that firstly the health personnel and then the people should be informed about the deadly parasites and the cleaning of the air conditions should be done in certain periods.

Production of a monoclonal antibody against a mannose-binding protein of Acanthamoeba culbertsoni and its localization

Amoebae from the genus Acanthamoeba are facultative pathogens of humans and other animals. In humans they most frequently infect the eye causing a sight threatening infection known as Acanthamoeba keratitis (AK), and also cause an often fatal encephalitis (GAE). A mannose-binding protein (MBP) has been identified as being important for Acanthamoeba infection especially in AK. This lectin has previously been characterized from Acanthamoeba castellanii as consisting of multiple 130 kDa subunits. MBP expression correlates with pathogenic potential and is expressed in a number of Acanthamoeba species. Here we report the purification of a similar lectin from Acanthamoeba culbertsoni and the production of a monoclonal antibody to it. The A. culbertsoni MBP was isolated by affinity chromatography using α-D-mannose agarose and has an apparent molecular weight of 83 kDa. The monoclonal antibody is an IgM that is useful in both western blots and immunofluorescence. We expect that this antibody will be useful in the study of the pathology of A. culbertsoni and in its identification in clinical samples.

Nuclear Group I introns with homing endonuclease genes in Acanthamoeba genotype T4

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.

DNA extraction from amoebal isolates and genotype determination of Acanthamoeba from tap water in Latvia.

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.

Structural and functional studies of histidine biosynthesis in Acanthamoeba spp. demonstrates a novel molecular arrangement and target for antimicrobials.

Acanthamoeba is normally free-living, but sometimes facultative and occasionally opportunistic parasites. Current therapies are, by necessity, arduous and yet poorly effective due to their inabilities to kill cyst stages or in some cases to actually induce encystation. Acanthamoeba can therefore survive as cysts and cause disease recurrence. Herein, in pursuit of better therapies and to understand the biochemistry of this understudied organism, we characterize its histidine biosynthesis pathway and explore the potential of targeting this with antimicrobials. We demonstrate that Acanthamoeba is a histidine autotroph, but with the ability to scavenge preformed histidine. It is able to grow in defined media lacking this amino acid, but is inhibited by 3-amino-1,2,4-triazole (3AT) that targets Imidazoleglycerol-Phosphate Dehydratase (IGPD) the rate limiting step of histidine biosynthesis. The structure of Acanthamoeba IGPD has also been determined in complex with 2-hydroxy-3-(1,2,4-triazol-1-yl) propylphosphonate [(R)-C348], a recently described novel inhibitor of Arabidopsis thaliana IGPD. This compound inhibited the growth of four Acanthamoeba species, having a 50% inhibitory concentration (IC50) ranging from 250–526 nM. This effect could be ablated by the addition of 1 mM exogenous free histidine, but importantly not by physiological concentrations found in mammalian tissues. The ability of 3AT and (R)-C348 to restrict the growth of four strains of Acanthamoeba spp. including a recently isolated clinical strain, while not inducing encystment, demonstrates the potential therapeutic utility of targeting the histidine biosynthesis pathway in Acanthamoeba.

Effect of ethanol pretreatment in Acanthamoeba keratitis: a long-term follow-up study.

PurposeThe aim of this study was to evaluate the long-term outcomes of ethanol pretreatment in Acanthamoeba keratitis (AK).Patients and methodsThis single-center, retrospective, interventional study included 22 patients (24 eyes) who developed AK and underwent ethanol pretreatment between 2009 and 2015. Samples for smears, polymerase chain reaction, and culture for evidence of Acanthamoeba were collected. After ethanol pretreatment, the patients were treated with corneal epithelial debridement, topical 0.02% polyhexamethylene biguanide, and 0.1% propamidine isethionate. The primary outcomes were a clinically stable ocular surface, complete recovery from corneal infection, and acceptable corneal haze. The secondary outcome measure was improvement in best-corrected visual acuity. Complications and predictors of the visual outcome were also recorded.ResultsEthanol pretreatment was successful in 20 (83.3%) of the 24 eyes, and no further optical keratoplasty was required. Four eyes required rescue therapeutic keratoplasty because of rapid progression of AK. Patients in whom ethanol pretreatment was successful achieved good final visual outcomes regardless of sex, age, or causative Acanthamoeba species. Patients with worse initial best-corrected visual acuity and rigid gas permeable lens-related AK had better improvement in vision.ConclusionEthanol as a pretreatment for AK is safe and effective. Combined with corneal epithelial debridement, ethanol pretreatment may preclude the need for optical and therapeutic keratoplasty. This technique is suitable for all stages of AK presenting within 3 weeks of symptom onset and achieves favorable results especially in early AK.

Deciphering the genomes of 16 Acanthamoeba species does not provide evidence of integration of known giant virus-associated mobile genetic elements

Giant viruses infect protozoa, especially amoebae of the genus Acanthamoeba. These viruses possess genetic elements named Mobilome. So far, this mobilome comprises provirophages which are integrated into the genome of their hosts, transpovirons, and Maverick/Polintons. Virophages replicate inside virus factories within Acanthamoeba and can decrease the infectivity of giant viruses. The virophage infecting CroV was found to be integrated in the host of CroV, Cafeteria roenbergensis, thus protecting C. roenbergensis by reduction of CroV multiplication. Because of this unique property, assessment of the mechanisms of replication of virophages and their relationship with giant viruses is a key element of this investigation. This work aimed at evaluating the presence and the dynamic of these mobile elements in sixteen Acanthamoeba genomes. No significant traces of the integration of genomes or sequences from known virophages were identified in all the available Acanthamoeba genomes. These results brought us to hypothesize that the interactions between mimiviruses and their virophages might occur through different mechanisms, or at low frequency. An additional explanation could be that our knowledge of the diversity of virophages is still very limited.

Confocal autofluorescence identification of bacteria, fungi, and acanthamoeba in infected porcine cornea models

Current standard in the clinical diagnostic of corneal infection in vivo is reflectance confocal microscopy using photons in the wavelength range of 600 s nm. While pathogens with large spatial features (∼10 μm) such as acanthamoeba and fungal species can be identified, smaller pathogens such as bacteria (∼1 μm) are difficult to detect. Furthermore, reflected light produces images anywhere there are index of refractive index changes. The non-specificity of such images can contribute to confusion in pathogen identification. In this work, we used 488 nm to excite autofluorescence in nine common pathogens (4 bacteria, 3 fungi, and 2 acanthamoeba species) inoculated in ex vivo porcine corneas. We found that the detected green autofluroescence is sufficient to clearly image the pathogens. Histograms of the autofluorescence intensity can also act as a metric in pathogen classification.

Development and optimization of new culture media for Acanthamoeba spp. (Protozoa: Amoebozoa)

The isolation and growth in axenic liquid media of Acanthamoeba strains is necessary in order to carry out primary in vitro drug screening. Amoebic isolates which are hard to grow in the current liquid media have been reported. Such circumstances hampers the ability of conducting drug sensitivity tests. Therefore, finding suitable universal growth media for Acanthamoeba species is required. The present study was aimed at the development of liquid medium suitable for growing a fastidious (F) genotype T3 Acanthamoeba isolate, and eventually for other genotypes of this genus as well. Trophozoite growth was indirectly monitored by respiration analysis with oxygen-sensitive microplates (OSM) and further confirmed by manual counting. Media were empirically designed and tested first in a non-fastidious (NF) T3 isolate and then tested with 14 different strains, including the fastidious one. Combinations of nutritive components such as meat/vegetable broth, LB medium, malt and skimmed milk led to the design of new media suitable for culturing all the isolates tested, in conditions similar to those obtained in standard culture media such as PYG or CERVA.

Molecular Identification and Genotyping of Acanthamoeba Isolated from Environmental Sources in Kashan, Central Iran

Background: Acanthamoeba is one of the most common opportunistic free-living amoebae, with ubiquitous presence in various en- vironmental sources. Pathogenic strains of Acanthamoeba are the causative agents of amoebic keratitis and granulomatous amoebic encephalitis. Objectives: The aim of the present study was to identify Acanthamoeba genotypes in soil, hospital dust, and stagnant water samples from Kashan, Central Iran. Methods: In this cross sectional study, a total of 122 samples from soil (n, 32), hospital dust (n, 40), and stagnant water (n, 50) were collected and examined for the presence of free-living amoebae and Acanthamoeba species. All the samples were cultured onto non-nutrient agar plates for detection of free-living amoebae. Acanthamoeba species was identified by polymerase chain reaction (PCR) assay, using specific primers. A total of 29 Acanthamoeba isolates were sequenced, and different genotypes were detected via sequence analysis. Results: The results showed that 82.8% (101/122) of samples were positive for free-living amoebae. The PCR assay revealed that 62.5%, 52.5%, and 50% of soil, hospital dust, and stagnant water samples were positive for Acanthamoeba species, respectively. Moreover, T4, T5, T2, T7, and T11 genotypes were identified. The most common genotype was T4 (76%), isolated from stagnant water. Conclusions: Acanthamoeba is a prevalent species in the soil, hospital dust, and stagnant water of Kashan. As this protozoon can cause severe infections, health education and improvement of sanitation services are recommended for prevention of infection. Keywords: Acanthamoeba, Soil, Hospital Dust, Stagnant Water, Genotype, Iran

A new strategy for enhancing acanthamoebicidal activity with synthesis of nanoflower of Laurocerausus officinalis Roemer (cherry laurel) fruit extracts

Pathogenic Acanthamoeba species often cause infection known as Acanthamoeba keratitis among people who use contact lenses. It is a type of infection that can result in corneal ulceration, visual loss or even blindness, if not treated. There are various therapeutic options available in the treatment of Acanthamoeba infections but they are usually tough treatments with limited efficacy. For instance, hydrogen peroxide (H2O2) is a commonly used contact lens disinfectant which is effective against Acanthamoeba but it is toxic to the cornea. For these reasons, new and more efficacious treatment options are required for Acanthamoeba infections. In this context, plants are considered natural resources for the discovery of new drugs. Laurocerasus officinalis Roem. (cherry laurel) (Rosaceae) grows in Black Sea region; and it is known as "Taflan", "Laz kirazı" or "Karayemis". Local people are using the seeds against diabetes, while the fruits are consuming as food, and used fordiuretic and passing kidney stones. It has also been reported that the seeds of the cherry laurel are used as an antiparasitic agent in this area. The aim of the study was to confirm the traditionally use of antiparasitic activity of this fruit and to increase the potential effect by means of organic-inorganic hybrid synthesis. Total phenol contents of methanol extracts prepared from endocarp, mesocarp and seeds of the fruit were calculated. The effects of methanol extracts and nano flower (NFs) plants synthesized from these extracts on the proliferation of Acanthamoeba castellanii were investigated. Thus, for the first time, novel organic-inorganic nanobio-antiparasitic agents called NFs were produced from cherry laurel and the increase in the amoebicidal activity of the NFs was elucidated. The characterization of NFs were determined with Scanning Electron Microscopy (SEM), Fourier Transform Infrared Spectrometer (FT-IR) and Energy-Dispersive X-ray (EDX) techniques. In addition, the catalytic activity of the fruit extracts and the NFs were measured against guaiacol in the presence of H2O2. The viability testing of A.castellanii cysts used for amoebicidal activity was performed using 4% trypan blue. Methanol extracts and nano-flowers were prepared at concentrations of 32, 16, 8, 4, 2 and 1 mg/ml in 0.9% saline and distributed 200 μl each in tubes and incubated in the room temperature with the addition of 200 μl of 98% viable A.castellani parasites. The results were evaluated using the SPSS V.22.0 program and it was determined that there was a significant increase in the amoebicidal activity of NFs compared with the other extracts according to variance analysis (p≤ 0.05). In the study, it was determined that samples killed parasites or reduced parasite proliferation at certain times. As a result, NFs synthesized from fruit extracts were demonstrated about three times more effective than the non hybrid extracts for amoebicidal activity. This situation can be explained as high proliferative effect of a new nano-bio-antiparasitic agent known as nanoflower against A.castellanii.

Comparative examination on selected amphizoic amoebae in terms of their in vitro temperature tolerance – a possible indirect marker of potential pathogenicity of Acanthamoeba strains

Acanthamoeba species are ubiquitous in natural and man-made environments worldwide; some strains are able to colonize human eyes as facultative parasites. It has been shown that environmental and clinical isolates/species of Acanthamoeba vary in their pathogenicity. In this study we examine and compare the in vitro effects of the changing temperature on the population dynamics of subsequent amoebic strains. Identification of Acanthamoeba strain by morphological and molecular methods and temperature assays were performed. Monitoring of the corneal and environmental strains showed changes in population densities and a termo-tolerance correlating with pathogenicity of amoebae. Comparative assessment of results indicated differences in viability of amoebic populations in exponential growth phase in vitro cultivation. The increased awareness of the threat is needed for better understanding of impact of factors examined on pathogenesis in human infected with Acanthamoeba strains.