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Granulomatous amoebic encephalitis is a rare but likely under‐recognised form of subacute, usually fatal, encephalitis. We present an illustrative case report and literature review of Australian cases, summarising clinical features, diagnostic methods, treatment and outcomes. [ABSTRACT FROM AUTHOR]

Free‐living amoebae of the genus Acanthamoeba are infected by various bacteria in nature, and thus bacteria can protect themselves from adverse environmental conditions. Contrary to this ameba–bacteria relationship whether Acanthamoeba has antibacterial effects on bacteria is the different aspect of the relationship between these microorganisms. In this study, we investigate various Acanthamoeba strains have antibacterial effects on various Staphylococcus strains. Three environmental Acanthamoeba strains, isolated from various aquatic environments in Turkey, and Acanthamoeba castellanii ATCC 50373 standard strains were used in the study. The antistaphylococcal effect of cell‐free supernatant (CFS) obtained from these amoebae against 12 different Staphylococcus bacteria was investigated by colony counting method. In addition, the pathogenicity of the tested Acanthamoeba strains was determined using osmotolerance and thermotolerance tests. CFSs obtained from Acanthamoeba were found to have varying degrees of antistaphylococcal effects on various Staphylococcus strains (0%–100%). It was determined that the CFS of the standard Acanthamoeba strain showed 100% inhibitory effect against one clinical methicillin‐resistant Staphylococcus aureus strain (M2). Also, CFS of Ugöl strain showed 99.97% inhibitory effect against one clinical methicillin‐sensitive Staphylococcus epidermidis strain (L3). It was determined that all Acanthamoeba isolates had no pathogenic potential. According to the results, it has been observed that Acanthamoeba produces antibacterial substance(s) against Staphylococcus bacteria and that the ameba–bacteria relationship may also result in the detriment of the bacteria. Furthermore, the current study indicates that new and natural antimicrobial agents from Acanthamoeba can be used as an alternative to infections caused by Staphylococcus. [ABSTRACT FROM AUTHOR]

Following the discovery of Acanthamoeba polyphaga mimivirus, diverse giant viruses have been isolated. However, only a small fraction of these isolates have been completely sequenced, limiting our understanding of the genomic diversity of giant viruses. MinION is a portable and low‐cost long‐read sequencer that can be readily used in a laboratory. Although MinION provides highly error‐prone reads that require correction through additional short‐read sequencing, recent studies assembled high‐quality microbial genomes only using MinION sequencing. Here, we evaluated the accuracy of MinION‐only genome assemblies for giant viruses by re‐sequencing a prototype marseillevirus. Assembled genomes presented over 99.98% identity to the reference genome with a few gaps, demonstrating a high accuracy of the MinION‐only assembly. As a proof of concept, we de novo assembled five newly isolated viruses. Average nucleotide identities to their closest known relatives suggest that the isolates represent new species of marseillevirus, pithovirus and mimivirus. The assembly of subsampled reads demonstrated that their taxonomy and genomic composition could be analysed at the 50× sequencing coverage. We also identified a pithovirus gene whose homologues were detected only in metagenome‐derived relatives. Collectively, we propose that MinION‐only assembly is an effective approach to rapidly perform a genome‐wide analysis of isolated giant viruses. [ABSTRACT FROM AUTHOR]

Key Clinical Message: A simple culturing method for Acanthamoeba available at a clinical laboratory is a key for making timely diagnosis and starting treatment with topical 0.02% chlorhexidine gluconate eye drops, together with 0.1% miconazole or fluconazole eye drops. A 19‐year‐old woman with pain and injection in the right eye showed spotty corneal infiltration and radiating linear opacity. Suspicious of Acanthamoeba keratitis, corneal scraping, and the soft contact lens were sent to in‐house clinical laboratory to culture successfully Acanthamoeba on Sabouraud dextrose agar plate painted with heat‐treated dead bacilli. [ABSTRACT FROM AUTHOR]

Summary: The environmental bacterium Legionella pneumophila causes the pneumonia Legionnaires' disease. The opportunistic pathogen forms biofilms and employs the Icm/Dot type IV secretion system (T4SS) to replicate in amoebae and macrophages. A regulatory network comprising the Legionella quorum sensing (Lqs) system and the transcription factor LvbR controls bacterial motility, virulence and biofilm architecture. Here we show by comparative proteomics that in biofilms formed by the L. pneumophila ΔlqsR or ΔlvbR regulatory mutants the abundance of proteins encoded by a genomic 'fitness island', metabolic enzymes, effector proteins and flagellar components (e.g. FlaA) varies. ∆lqsR or ∆flaA mutants form 'patchy' biofilms like the parental strain JR32, while ∆lvbR forms a 'mat‐like' biofilm. Acanthamoeba castellanii amoebae migrated more slowly through biofilms of L. pneumophila lacking lqsR, lvbR, flaA, a functional Icm/Dot T4SS (∆icmT), or secreted effector proteins. Clusters of bacteria decorated amoebae in JR32, ∆lvbR or ∆icmT biofilms but not in ∆lqsR or ∆flaA biofilms. The amoeba‐adherent bacteria induced promoters implicated in motility (PflaA) or virulence (PsidC, PralF). Taken together, the Lqs‐LvbR network (quorum sensing), FlaA (motility) and the Icm/Dot T4SS (virulence) regulate migration of A. castellanii through L. pneumophila biofilms, and – apart from the T4SS – govern bacterial cluster formation on the amoebae. [ABSTRACT FROM AUTHOR]

In vivo confocal microscopy (IVCM) is a relatively new ocular imaging technique that permits morphological and quantitative assessment of the living cornea on the cellular level. The applications for IVCM in clinical ophthalmology are numerous and diverse. There are several advantages inherent to IVCM over standard diagnostic techniques currently used to confirm a diagnosis of infectious keratitis in veterinary ophthalmology. With IVCM, images can be viewed in real‐time providing immediate diagnostic information. Traumatic corneal sampling techniques are avoided, and the procedure can be repeated as frequently as is clinically indicated without risk of corneal tissue damage. Both superficial and deep corneal lesions can be evaluated by IVCM in an atraumatic fashion. Microorganism viability is not required for their detection and specialized diagnostic laboratory assay procedures are not necessary. Many larger infectious agents can be directly identified within corneal lesions by IVCM, including fungi and parasites such as Acanthamoeba spp. In other situations, such as bacterial infectious crystalline keratopathy, the biological systems associated with the microorganism can be detected within the cornea. The current resolution of IVCM is inadequate to directly visualize some corneal infectious agents, such as herpesviruses, but host responses and virus‐infected epithelial cells can be identified. This review summarizes the current knowledge and applications of IVCM in the management of infectious keratitis in veterinary ophthalmology, including its use in animals with bacterial, fungal, parasitic, and viral keratitis. [ABSTRACT FROM AUTHOR]

Actophorin, which was recently tested for crystallization under microgravity on the International Space Station, was subjected to mutagenesis to identify a construct with improved biophysical properties that were expected to improve the extent of diffraction. First, 20 mutations, including one C‐terminal deletion of three residues, were introduced individually into actophorin, resulting in modest increases in thermal stability of between +0.5°C and +2.2°C. All but two of the stabilizing mutants increased both the rates of severing F‐actin filaments and of spontaneous polymerization of pyrenyl G‐actin in vitro. When the individual mutations were combined into a single actophorin variant, Acto‐2, the overall thermal stability was 22°C higher than that of wild‐type actophorin. When an inactivating S2P mutation in Acto‐2 was restored, Acto‐2/P2S was more stable by 20°C but was notably more active than the wild‐type protein. The inactivating S2P mutation reaffirms the importance that Ser2 plays in the F‐actin‐severing reaction. The crystal structure of Acto‐2 was solved to 1.7 Å resolution in a monoclinic space group, a first for actophorin. Surprisingly, despite the increase in thermal stability, the extended β‐turn region, which is intimately involved in interactions with F‐actin, is disordered in one copy of Acto‐2 in the asymmetric unit. These observations emphasize the complex interplay among protein thermal stability, function and dynamics. [ABSTRACT FROM AUTHOR]

Acanthamoeba is a genus of free‐living amoebae, pervasively found in the environment. Most of its pathogenic species are the causative agent of sight‐threatening Acanthamoeba keratitis and fatal granulomatous amoebic encephalitis. Despite the advancements in the field of chemotherapy, treating Acanthamoeba infections is still challenging due to incomplete knowledge of the complicated pathophysiology. In case of infection, the treatment regimen for the patients is often ineffective due to delayed diagnosis, poor specificity, and side‐effects. Besides the resistance of Acanthamoeba cysts to most of the drugs, the recurrence of infection further complicates the recovery. Thus, it is necessary to develop an effective treatment which can eradicate these rare, but serious infections. Based on various computational and in vitro studies, it has been established that the synthetic scaffolds such as heterocyclic compounds may act as potential drug leads for the development of antiamoebic drugs. In this review, we report different classes of synthetic compounds especially heterocyclic compounds which have shown promising results against Acanthamoeba. Moreover, the antiamoebic activities of synthetic compounds with their possible mode of actions against Acanthamoeba, have been summarized and discussed in this review. [ABSTRACT FROM AUTHOR]

Objective: Heart transplant is one of the accepted treatments for some patients with advanced heart failure. Of note, transplant surgeries may cause different infections and complications for patients during the post‐transplant period. A wide variety of opportunistic organisms caused these infections including bacteria, fungi, viruses, and protozoa particularly Free‐living amoebae (FLA). This study aims to study the presence of pathogenic FLA from the oral cavity of post‐heart transplant recipients. Methods: Throat swabs were collected from 80 patients who underwent post‐heart transplant surgery. All swabs were immediately cultured in non‐nutrient agar (2%). PCR and sequencing of 18S rRNA gene (DF3 region) of Acanthamoeba isolates were performed using genus‐specific primers. Genetic associations among sequenced genotypes inferred by the 18S rRNA gene obtained by MEGA X and a phylogenetic tree were constructed using the maximum likelihood algorithm and Kimura 2‐parameter model. Results: Out of 80 samples collected from post‐heart transplant patients, six (7.5%) samples showed positive outgrowth of Acanthamoeba based on the page key and sequencing of the DF3 region. Sequence similarity of ASA1 by basic local alignment search tool(n) showed that five isolates (ANHT1, ANHT2, ANHT3, ANHT4, and ANHT5) belonged to Acanthamoeba T5 genotype corresponding to A. lenticulata and one strain (ANHT6) belonged to the T4 genotype. Conclusion: To the best of our knowledge for the first time, a comprehensive study of Acanthamoeba genotypes isolated from throat samples of heart transplant recipients is described. Heart transplantation patients can be colonized by FLA and are therefore at risk of developing an invasive infection. Physicians' awareness of central nervous system infections related to FLAs and preventive and control measures of patients with compromised immune status due to heart transplant surgery are of utmost importance. [ABSTRACT FROM AUTHOR]

Actophorin, a protein that severs actin filaments isolated from the amoeba Acanthamoeba castellanii, was employed as a test case for crystallization under microgravity. Crystals of purified actophorin were grown under microgravity conditions aboard the International Space Station (ISS) utilizing an interactive crystallization setup between the ISS crew and ground‐based experimenters. Crystals grew in conditions similar to those grown on earth. The structure was solved by molecular replacement at a resolution of 1.65 Å. Surprisingly, the structure reveals conformational changes in a remote β‐turn region that were previously associated with actophorin phosphorylated at the terminal residue Ser1. Although crystallization under microgravity did not yield a higher resolution than crystals grown under typical laboratory conditions, the conformation of actophorin obtained from solving the structure suggests greater flexibility in the actophorin β‐turn than previously appreciated and may be beneficial for the binding of actophorin to actin filaments. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Purpose: Contact lenses have direct contact with the corneal surface and can induce sight‐threatening infection of the cornea known as Acanthamoeba keratitis. The objective of this study was to evaluate the dysprosium‐based nanoparticles (Dy‐based NPs), namely Fe3O4‐PEG‐Dy2O3 nanocomposites and Dy(OH)3 nanorods, as an active component against Acanthamoeba sp., as well as the possibility of their loading onto contact lenses as the drug administering vehicle to treat Acanthamoeba keratitis (AK). Methods: The Dy‐based NPs were synthesized, and they were loaded onto commercial contact lenses. The loading content of the NPs and their release kinetics was determined based on the absorbance of their colloidal solution before and after soaking the contact lenses. The cytotoxicity of the NPs was evaluated, and the IC50 values of their antiamoebic activity against Acanthamoeba sp. were determined by MTT colorimetric assay, followed by observation on the morphological changes by using light microscopy. The mechanism of action of the Dy‐based NPs against Acanthamoeba sp. was evaluated by DNA laddering assays. Results: The loading efficiencies of the Dy‐based NPs onto the contact lens were in the range of 30.6–36.1% with respect to their initial concentration (0.5 mg ml–1). The Dy NPs were released with the flux approximately 5.5–11 μg cm–2 hr–1, and the release was completed within 10 hr. The emission of the NPs consistently showed a peak at 575 nm due to Dy3+ ion, offering the possible monitoring and tracking of the NPs. The SEM images indicated the NPs are aggregated on the surface of the contact lenses. The DNA ladder assay suggested that the cells underwent DNA fragmentation, and the cell death was due most probably to necrosis, rather than apoptosis. The cytotoxicity assay of Acanthamoeba sp. suggested that Fe3O4‐PEG, Fe3O4‐PEG‐Dy2O3, Dy(NO3)3.6H2O and Dy(OH)3 NPs have an antiamoebic activity with the IC50 value being 4.5, 5.0, 9.5 and 22.5 μg ml–1, respectively. Conclusions: Overall findings in this study suggested that the Dy‐based NPs can be considered as active antiamoebic agents and possess the potential as drugs against Acanthamoeba sp. The NPs could be loaded onto the contact lenses; thus, they can be potentially utilized to treat Acanthamoeba keratitis (AK). [ABSTRACT FROM AUTHOR]

Acanthamoeba keratitis is an infection caused by a unicellular protozoan of the genus Acanthamoeba that is universally widespread. Until now, most cases were reported in contact lens wearers, although it is also a reality for non‐wearers, mostly connected to corneal trauma. There is also a variation in incidence regarding the aetiology of the disease between developed and developing countries. Purpose: This work is based on a literature review, and the main goal is to deepen the knowledge about Acanthamoeba keratitis, presenting the main risk factors and focusing on prevention actions for this type of corneal infection since the treatments are not always effective. It targets specialists in visual health to strengthen their knowledge in this area, as well as to allow them to better inform their patients about hygiene care, appropriate measures of disinfection and ways to minimise the risk of infection. At this stage, it is important to highlight the essential role that practitioners play in fitting, monitoring and following‐up patients to minimise the danger of infection. Recent findings: It is well recognised that corneal trauma facilitates invasion by leaving an open door for microorganisms to penetrate the cornea. In addition to trauma, risk factors are mostly associated with patients' behaviours, such as interaction of contact lenses with contaminated water in the shower, swimming pools and beaches, etc., lack of hygiene habits with contact lenses and respective cases, and the use of ineffective disinfecting solutions. The fact that a disinfecting solution is not completely effective against trophozoites and/or cysts, both forms of Acanthamoeba's lifecycle, can cause the infection since one cyst alone leads to the emergence of a whole new population of Acanthamoeba. Summary: It is necessary to reduce the risk of infection and, beyond the need to promote patient education to encourage correct CL hygiene behaviours, it should also be highlighted that there is an urgent need to enhance the efficacy of CL disinfection systems against all strains and both stages of Acanthamoeba through the creation of standardised methods. The ease of purchasing CLs without any supervision must also be considered a concern, and, in the near future, it is also important to develop and implement effective diagnostic methods and treatments for Acanthamoeba keratitis. [ABSTRACT FROM AUTHOR]

Acanthamoeba spp. cause a corneal infection, Acanthamoeba keratitis (AK), and a cerebral infection, granulomatous amoebic encephalitis (GAE). Though aggressive chemotherapy has been able to kill the active trophozoite form of Acanthamoeba, the encysted form of this parasite has remained problematic to resist physiological concentrations of drugs. The emergence of encysted amoeba into active trophozoite form poses a challenge to eradicate this parasite. Acanthamoeba trophozoites have active metabolic machinery that furnishes energy in the form of ATPs by subjecting carbohydrates and lipids to undergo pathways including glycolysis and beta‐oxidation of free fatty acids, respectively. However, very little is known about the metabolic preferences and dependencies of an encysted trophozoite on minerals or potential nutrients that it consumes to live in an encysted state. Here, we investigate the metabolic and nutrient preferences of the encysted trophozoite of Acanthamoeba castellanii and the possibility to target them by drugs that act on calcium ion dependencies of the encysted amoeba. The experimental assays, immunostaining coupled with bioinformatics tools show that the encysted Acanthamoeba uses diverse nutrient pathways to obtain energy in the quiescent encysted state. These findings highlight potential pathways that can be targeted in eradicating amoebae cysts successfully. [ABSTRACT FROM AUTHOR]

This study developed dose response models for determining the probability of eye or central nervous system infections from previously conducted studies using different strains of Acanthamoeba spp. The data were a result of animal experiments using mice and rats exposed corneally and intranasally to the pathogens. The corneal inoculations of Acanthamoeba isolate Ac 118 included varied amounts of Corynebacterium xerosis and were best fit by the exponential model. Virulence increased with higher levels of C. xerosis. The Acanthamoeba culbertsoni intranasal study with death as an endpoint of response was best fit by the beta‐Poisson model. The HN‐3 strain of A. castellanii was studied with an intranasal exposure and three different endpoints of response. For all three studies, the exponential model was the best fit. A model based on pooling data sets of the intranasal exposure and death endpoint resulted in an LD50 of 19,357 amebae. The dose response models developed in this study are an important step towards characterizing the risk associated with free‐living amoeba like Acanthamoeba in drinking water distribution systems. Understanding the human health risk posed by free‐living amoeba will allow for quantitative microbial risk assessments that support building design decisions to minimize opportunities for pathogen growth and survival. [ABSTRACT FROM AUTHOR]

A series of phosphonium amphiphilic compounds was synthesized. Cationic parts of molecules contain triphenylphosphonium moieties. Lipophilic parts of compounds are represented by straight alkyl chain or the alkyl chains which are ornamented by benzyl or metronidazole. The physicochemical properties of phosphonium amphiphilic compounds were investigated by the measurements of surface tension and conductivity. The critical micelle concentration (cmc), the surface tension value at the cmc (γcmc), the surface area at the surface saturation per head group (Acmc) were determined. The lowest cmc value was determined for phosphonium salts with straight dodecyl alkyl chain. Its value was 1.5 × 10−3 mol dm−3. Surface tension at the cmc decreases with the addition of bulky moieties (benzyl, radical from metronidazol) at the end of alkyl chains. Biological activities of compounds were studied on human erythrocytes and strains of Acanthamoeba lugdunensis and Acanthamoeba quina. Dodecyltriphenylphosphonium bromide showed the highest activity against Acanthamoeba. To the best of our knowledge, it is the first compound of the group of phosphonium amphiphiles, which exhibited high activity against Acanthamoeba. The determined structure–activity relationship indicated nonspecific trophocidal and hemolytic activity that depends on physicochemical properties of the studied compounds. [ABSTRACT FROM AUTHOR]

The distribution pattern of the meiotic machinery in known eukaryotes is most parsimoniously explained by the hypothesis that all eukaryotes are ancestrally sexual. However, this assumption is questioned by preliminary results, in culture conditions. These suggested that Acanthamoeba, an organism considered to be largely asexual, constitutively expresses meiosis genes nevertheless—at least in the lab. This apparent disconnect between the "meiosis toolkit" and sexual processes in Acanthamoeba led to the conclusion that the eukaryotic ancestor is asexual. In this review, the "meiosis toolkit" is rigorously defended, drawing on numerous research articles. Additionally, the claim of constitutive meiotic gene expression is probed in Acanthamoeba via the same transcriptomics data. The results show that the expression of the meiotic machinery is not constitutive in Acanthamoeba as claimed before. Furthermore, it is argued that this would have no implications for understanding the nature of the eukaryotic ancestor, regardless of the result. [ABSTRACT FROM AUTHOR]

Purpose: Acanthamoeba (AA) keratitis is a rare and severe infection with poor prognosis. The aim was to investigate the incidence and risk factors of AA keratitis in a large tertiary ophthalmology department in Denmark. Methods: A search was performed in our electronic patient records by the keywords: 'PHMB/polyhexanid', 'Brolene' or 'amoeba and chlorhexidine' from year 1994 to 2018, and afterwards medical records were reviewed. A total of 65 cases of AA keratitis were hereby identified. Results: Patients were relatively young, median (range) age of 38 (15–70) years. A significant increase of diagnosing AA keratitis occurred from 0.13 cases per million per year in the first 5 years to 2.7 cases per million per year the last 5 years. Eighty‐nine per cent of patients were contact lens users, and 49% had received corticosteroids before the diagnosis was established. Severe pain was present in 34% of patients. Prognosis was poor with final visual acuity of logMAR (mean, 95% CI) 0.30 (0.18–0.41), 18% had transplantation à chaud, and 2% were enucleated. Conclusion: The study indicates that the incidence of AA keratitis is increasing, also in Denmark. Most patients were young contact lens users. The course of the disease is long and often painful, and the prognosis is poor despite relevant treatment. [ABSTRACT FROM AUTHOR]

Granulomatous amoebic encephalitis caused by free‐living amoebae is a rare condition that is difficult to diagnose and hard to treat, generally being fatal. Anti‐amoebic treatment is often delayed because clinical signs and symptoms may hide the probable causing agent misleading the appropriate diagnostic test. There are four genera of free‐living amoeba associated with human infection, Naegleria, Acanthamoeba sp., Balamuthia and Sappinia. Two boys were admitted with diagnosis of acute encephalitis. The history of having been in contact with swimming pools and rivers, supports the suspicion of an infection due to free‐living amoebae. In both cases a brain biopsy was done, the histology confirmed granulomatous amoebic encephalitis with the presence of amoebic trophozoites. [ABSTRACT FROM AUTHOR]

Summary: Background: Sporotrichosis is a group of zoonotic subcutaneous mycoses, found worldwide and caused by fungi belonging to the genus Sporothrix. Protozoans of the genus Acanthamoeba are widely distributed, and some species may be pathogenic and/or opportunistic. These organisms coexist in the same environment and may interact. Objectives: This study determined the profile of interactions of S schenckii sensu stricto and S brasiliensis with A castellanii, using an in vitro co‐culture model to evaluate the intrinsic characteristics of the two Sporothrix species and A castellanii. Methods: We compared the rate of phagocytosis of S schenckii sensu stricto and S brasiliensis by A castellanii; the viability of S schenckii sensu stricto and S brasiliensis after contact with A castellanii; the viability of the amoeba after contact with a fungal species; and the influence of S schenckii sensu stricto and S brasiliensis on the encystment process of A castellanii. Results: The analyses indicated that A castellanii phagocytised both S schenckii and S brasiliensis, with significantly more S schenckii than S brasiliensis in the first two hours of contact. Our results showed a significant increase in conidia and hyphae count after 72 hours of co‐culture of A castellanii with S brasiliensis, and the amoebae lysed after they ingested the fungi, indicating that the fungi probably used the amoebae as a source of nutrition. Conclusions: Our results were obtained in vitro and these organisms may not behave similarly in vivo; in vivo studies of co‐infections are necessary in order to gain a thorough understanding of this relationship. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Aims/Purpose: Novel effective medical therapy for Acanthamoeba Keratitis. Methods: Interventional observational case series. Compassionate use of Polihexanide 0.8 mg/mL in 2 Acanthamoeba keratits (AK) patients. Two cases of diagnosed Acanthamoeba keratitis were unsuccessfully treated with Poliexamidine biguanide 0.02% and propamidine 0.1% applied hourly then tapered weekly. Clinical worsening was observed up to 4 weeks. Compassionate use of Polihexanide 0.8 mg/mL eyedrops was started. Results: Corneal infection healed completely after 1 month in first case, and after 2 months of therapy in the second case. Corneal opacities and neovascularization decreased slowly, while BCVA (best corrected visual acuity) slightly and progressively increased per 3 months, and no infection recurred. Conclusions: AK is a painful, long‐lasting (12 months) infection that drastically reduces a patient's quality of life. Polihexanide 0.8 mg/mL monotherapy lowers illness duration, chance of recurrence, and need for surgery in high risk settings, improving visual outcomes and quality of life. [ABSTRACT FROM AUTHOR]

Over 600 cases of amoebic encephalitis caused by pathogenic free‐living amoebas (Balamuthia mandrillaris, Acanthamoeba spp., and Naegleria fowleri) have been reported worldwide, and in Japan, 24 cases have been reported from the first case in 1976 up to 2018. Among these cases, 18 were caused by B. mandrillaris, four by Acanthamoeba spp., one by N. fowleri, and one was of unknown etiology. Additionally, eight cases were diagnosed with encephalitis due to pathogenic free‐living amoebas before death, but only three cases were successfully treated. Unfortunately, all other cases were diagnosed by autopsy. These facts indicate that an adequate diagnosis is difficult, because encephalitis due to pathogenic free‐living amoebas does not show typical symptoms or laboratory findings. Moreover, because the number of cases is limited, other cases might have been missed outside of those diagnosed by autopsy. Cases of encephalitis caused by B. mandrillaris have been reported from all over Japan, with B. mandrillaris recently isolated from soil in Aomori prefecture. Therefore, encephalitis caused by pathogenic free‐living amoebas should be added to the differential diagnosis of encephalitis patients. [ABSTRACT FROM AUTHOR]

Shigella species and Acanthamoeba castellanii share the same ecological niches, and their interaction has been addressed in a limited number of research. However, there are still uncertain aspects and discrepant findings of this interaction. In the present study, the effects of the bacterial growth phase, cocultivation temperature and the type of culture media on the interaction of A. castellanii with Shigella dysenteriae, Shigella sonnei and Shigella flexneri were evaluated. In nutrient‐poor page's amoeba saline (PAS) medium, the number of recovered bacteria and the uptake rates were significantly higher in stationary phase cells than logarithmic phase cells. However, no significant differences were observed in the number of recovered bacteria and the uptake rates between logarithmic and stationary phase cells in nutrient‐rich peptone–yeast extract–glucose (PYG) medium. While the number of recovered bacteria was significantly higher in nutrient‐rich than nutrient‐poor media, in all the three Shigella species, the bacterial uptake rates were significantly higher in nutrient‐poor than nutrient‐rich media at both cocultivation temperatures. In both nutrient‐poor and nutrient‐rich media and at both cocultivation temperatures, the number of viable Shigella species after 24 h incubation were not influenced by the presence of A. castellanii. Although Shigella species did not proliferate in A. castellanii trophozoites, a considerable number of bacteria were survived in the trophozoites up to 15 days. From the public health perspective, the results of this study are important for further understanding of the nature of the interaction of these organisms and to deal with Shigella species in the environment. [ABSTRACT FROM AUTHOR]

Amoebae, bacteria and fungi containing biofilms occuring in the built environment Microbial investigations of various organic black stains from water systems or connected facilities revealed different constituting microorganisms. Surprisingly regularly, free‐living amoebae (FLA), especially of the genus Acanthamoeba, together with bacteria and black yeasts were found. Altogether we recognized a potentially three‐fold hygienic load emanating from the investigated biofilms which are potentially pathogenic amoebae, the likely involvement of pathogenic bacteria and the presence of opportunistic fungi. Furthermore we realized that the conditions at which such biofilms developed are characterized by a typical combination of environmental factors. These are high temperature, potentially intermittent wetness and connected water stress or high osmotic pressure/salinity and the application of detergents and occassionally disinfectants. [ABSTRACT FROM AUTHOR]

Abstract: Importance: Acanthamoeba is an increasingly prevalent cause of vision‐threatening microbial keratitis. Background: To assess the incidence, clinical presentation, diagnosis and outcomes of patients with Acanthamoeba keratitis (AK) in Auckland, New Zealand over a 7‐year period. Design: Retrospective observational consecutive case series. Participants: Fifty‐eight eyes of 52 patients diagnosed with AK. Methods: All cases of AK were identified using a cross‐referenced search of clinical, laboratory and pharmacy records from March 2009 to May 2016. Main Outcome Measures: Demographic and clinical data were collected including age, gender, risk factors, clinical manifestations, initial diagnosis, diagnostic investigations, treatment, presenting and final visual acuity and surgical interventions. Results: Contact lens (CL) use was noted in 96% of unilateral and 100% of bilateral cases. The mean duration of symptoms at presentation was 21 days and the mean duration from presentation to definitive diagnosis was 14 days. Initial diagnosis was recorded as CL‐related keratitis in 70.6%, viral keratitis in 15.5% and AK in 12.0%. The diagnosis was confirmed with In vivo confocal microscopy (IVCM) in 67.2%, corneal scrape in 22.4%, corneal biopsy in 1.7% and clinically in 8.6%. IVCM sensitivity was 83.0%. Surgical intervention was required in four patients, all with delayed diagnosis (range 63–125 days). The incidence of AK has more than doubled when compared with the preceding 7‐year period. Conclusions and Relevance: AK is a rare vision‐threatening protozoal infection with rapidly‐increasing incidence in New Zealand, predominantly affecting CL users. Diagnosis is often challenging and when delayed is associated with worse outcomes. IVCM offers rapid diagnosis with high sensitivity. [ABSTRACT FROM AUTHOR]

Summary: Soil‐borne amoeba Acanthamoeba S13WT has an endosymbiotic relationship with an environmental Neochlamydia bacterial strain. However, regardless of extensive experiments in liquid media, the biological advantage of the symbiosis remained elusive. We therefore explored the role of the endosymbiont in predator‐prey interactions on solid media. A mixed culture of the symbiotic or aposymbiotic amoebae and GFP‐expressing Escherichia coli or Salmonella Enteritidis was spotted onto the centre of a LB or B‐CYE agar plate preinoculated with a ring of mCherry‐expressing Legionella pneumophila (Legionella ‘wall’). The spread of the amoebae on the plate was assessed using a fluorescence imaging system or scanning electron microscopy. As a result, in contrast to the aposymbiotic amoebae, the symbiotic amoebae backpacked these GFP‐expressing bacteria and formed flower‐like fluorescence patterns in an anticlockwise direction. Other bacteria (Pseudomonas aeruginosa and Stenotrophomonas maltophilia), but not Staphylococcus aureus, were also backpacked by the symbiotic amoebae on LB agar, although lacked the movement to anticlockwise direction. Furthermore, in contrast to the aposymbiotic amoebae, the symbiotic amoebae backpacking the E. coli broke through the Legionella ‘wall’ on B‐CYE agar plates. Thus, we concluded that Acanthamoeba S13WT required the Neochlamydia endosymbiont to backpack human pathogenic bacteria and resist Legionella infection on solid agar. [ABSTRACT FROM AUTHOR]

Abstract: Acanthamoeba infections are difficult to diagnose and treat. We present a renal transplant patient who developed Acanthamoeba endophthalmitis on therapy with posaconazole and miltefosine for cutaneous acanthamobiasis. The patient was maintained on intracameral voriconazole injections, and oral azithromycin, fluconazole, and flucytosine. This case highlights novel presentations and treatments for acanthamoebic infection. [ABSTRACT FROM AUTHOR]

Abstract: Improved understanding of the mechanism of nutrient's uptake can enable targeted manipulation of nutrient sensing pathways in medically important pathogens to a greater degree than is currently possible. In this context, we present the use of spontaneous Raman microspectroscopy and coherent anti‐Stokes Raman spectroscopy to visualize the time‐dependent molecular interactions between the protozoan Acanthamoeba castellanii and host human cells. Human retinal pigment epithelial (ARPE‐19) cells were pre‐labelled with deuterated Phe (L‐Phe[D8]) and the uptake of the host derived L‐Phe(D8) by A. castellanii trophozoites was measured by Raman microspectroscopy for up to 48 hr post infection (hpi). This approach revealed a time‐dependent uptake pattern of this essential amino acid by A. castellanii trophozoites during the first 24 hpi with complete enrichment with L‐Phe(D8) detected in trophozoites at 48 hpi. In contrast, cell free A. castellanii trophozoites showed a modest uptake of only 16–18% L‐Phe(D8) from L‐Phe(D8)–supplemented culture medium after 3, 24, and 48 hr hpi. Coherent anti‐Stokes Raman spectroscopy microscopy was successfully used to monitor the reprogramming of lipids within the trophozoites as they engaged with host cells. The methodology presented here provides new advances in the ability to analyze the kinetic of amino acid acquisition by A. castellanii from host cell and extracellular environment, and to visualize lipid reprogramming within the trophozoite. [ABSTRACT FROM AUTHOR]

Acanthamoeba encephalitis is a rare, often fatal condition, particularly after HSCT, with 9 reported cases to date in the world literature. Our case was originally diagnosed with ALL at age 3 years, and after several relapses underwent HSCT at age 9 years. At 17 years of age, he was diagnosed with secondary AML for which he underwent a second allogeneic HSCT. He presented with acute-onset worsening neurological deficits on day +226 after the second transplant and a post-mortem diagnosis of Acanthamoeba encephalitis was established, with the aid of the CDC. [ABSTRACT FROM AUTHOR]

Placoderms (Devonian fossil fishes) are resolved phylogenetically to the base of jawed vertebrates and provide important evidence for evolutionary origins of teeth, particularly with respect to the Arthrodira. The arthrodires represent a derived group of placoderms; the dentition of other more primitive placoderms such as the acanthothoracids is less well known. Articulated acanthothoracid dental plates are rare; x-ray computed tomography of a single, unique specimen, along with 3D segmentation of bone, oral denticles and vascular spaces, provides intrinsic developmental and topological information relevant to tooth origins. Recently, a disarticulated element was identified as a dental plate of the acanthothoracid Romundina stellina, with synchrotron microtomography providing characters to comment on ongoing debates regarding the evolution of teeth. We used segmental quantitative methods to re-analyse this data, for comparison to the articulated and unquestionable acanthothoracid dental plates above. We demonstrate substantial differences between these, disputing the identity of the isolated plate of R. stellina as a dental plate, and thus its relevance to questions of tooth evolution. [ABSTRACT FROM AUTHOR]

Silver nanoparticles (AgNP) are used in a broad range of consumer products and industrial applications. During the regular product life cycle and disposal, AgNP are continuously released into the environment. Hence, the aim of this study was to investigate the potential ecotoxicological effects of AgNP exposure on amoebae. The Acanthamoeba castellanii ATCC 30234 strain and environmental isolate Acanthamoeba strain C5/2, which are both affiliated with genotype T4, were chosen as representatives of ecologically important soil protozoan organisms. The amoebae were exposed to citrate-stabilized AgNP (30 and 70 nm in size) for 24 h and 96 h at concentrations ranging from 600 µg L−1 to 20 mg L−1. A newly adopted cell culture based microscopic assay was applied to assess the adherence ability of the amoeba trophozoites. The general metabolic activity of Acanthamoeba was determined to be a second independent endpoint by means of intracellular reduction of the redox dye AlamarBlue®. The fate of AgNP within the amoebae and test solutions was visualized by light- and transmission electron microscopy (TEM). Both Acanthamoeba strains showed a significant dose-dependent decrease of adherence ability ( p < 0.04) and metabolic activity ( p < 0.01) after 96 h of AgNP exposure. The environmental Acanthamoeba strain C5/2 lost both its adherence ability and metabolic activity at lower AgNP concentrations than the type strain, indicating a higher sensitivity to ionic silver. This was confirmed by the application of AgNO3, provoking a higher effect level in strain C5/2. AgNP was visualized intracellularly by transmission electron microscopy within the cytoplasm of Acanthamoeba. This is the first report to show the ecotoxicological effects of short-term AgNP exposure on the soil protist Acanthamoeba, causing both changes in the adherence ability and metabolic activity of this amoeba. This combined approach may be a powerful tool in the future for predicting potential harmful ecotoxicological effects of AgNP exposure using soil protozoans. [ABSTRACT FROM AUTHOR]

Current treatments for Acanthamoeba keratitis are unspecific. Because of the presence of the resilient cyst form of the parasite, the infection is persistent. Silencing the key protein of cyst formation, glycogen phosphorylase, has shown potential for reducing encystment processes of the Acanthamoeba trophozoite. However, a suitable carrier to protect and deliver si RNA sequences is still needed. DOTAP: DOPE: DSPE- PEG liposomes were prepared by three different techniques and used to associate a therapeutic si RNA sequence. Liposomes prepared by film hydration followed by membrane extrusion were considered the most adequate ones with average size of 250 nm and zeta potential of +45 mV, being able to associate si RNA for at least 24 hr in culture medium. si RNA-liposomes could inhibit up to 66% of the encystment process. Cell viability studies demonstrated MTT reduction capacity higher than 80% after 3 hr incubation with this formulation. After 24 hr of incubation, LDH activity ranged for both the formulations from around 4% to 40%. In vivo tolerance studies in mice showed no macroscopic alteration in the eye structures up to 24 hr after eight administrations during 1 day. Histological studies showed regular tissue architecture without any morphological alteration. Overall, these results suggest that the formulations developed are a promising new strategy for the treatment of ocular keratitis caused by Acanthamoeba spp. [ABSTRACT FROM AUTHOR]

It is unclear how the water-based pathogen, Legionella pneumophila (Lp), and associated free-living amoeba ( FLA) hosts change or are changed by the microbial composition of drinking water ( DW) biofilm communities. Thus, this study characterized the bacterial community structure over a 7-month period within mature (> 600-day-old) copper DW biofilms in reactors simulating premise plumbing and assessed the impact of temperature and introduction of Lp and its FLA host, Vermamoeba vermiformis (Vv), co-cultures (LpVv). Sequence and quantitative PCR ( qPCR) analyses indicated a correlation between LpVv introduction and increases in Legionella spp. levels at room temperature ( RT), while at 37°C, Lp became the dominant Legionella spp. qPCR analysis suggested Vv presence may not be directly associated with Lp biofilm growth at RT and 37°C, but may contribute to or be associated with non-Lp legionellae persistence at RT. Two-way PERMANOVA and PCoA revealed that temperature was a major driver of microbiome diversity. Biofilm community composition also changed over the seven-month period and could be associated with significant shifts in dissolved oxygen, alkalinity and various metals in the influent DW. Hence, temperature, biofilm age, DW quality and transient intrusions/amplification of pathogens and FLA hosts may significantly impact biofilm microbiomes and modulate pathogen levels over extended periods. [ABSTRACT FROM AUTHOR]

Here we describe features of apoptosis in unicellular Acanthamoeba castellanii belonging to the T4 genotype. When exposed to apoptosis-inducing compounds such as doxorubicin, A. castellanii trophozoites exhibited cell shrinkage and membrane blebbing as observed microscopically, DNA fragmentation using agarose gel electrophoresis, and phosphatidylserine (PS) externalization using annexin V immunostaining. Overall, these findings suggest the existence of apoptosis in A. castellanii possibly mediated by intrinsic apoptotic cascade. Further research in this field could provide avenues to selectively induce apoptosis in A. castellanii by triggering intrinsic apoptotic cascade. [ABSTRACT FROM AUTHOR]

Disseminated acanthamoebiasis is a rare, often fatal, infection most commonly affecting immunocompromised patients. We report a case involving sinuses, skin, and bone in a 60-year-old woman 5 months after heart transplantation. She improved with a combination of flucytosine, fluconazole, miltefosine, and decreased immunosuppression. To our knowledge, this is the first case of successfully treated disseminated acanthamoebiasis in a heart transplant recipient and only the second successful use of miltefosine for this infection among solid organ transplant recipients. Acanthamoeba infection should be considered in transplant recipients with evidence of skin, central nervous system, and sinus infections that are unresponsive to antibiotics. Miltefosine may represent an effective component of a multidrug therapeutic regimen for the treatment of this amoebic infection. [ABSTRACT FROM AUTHOR]

The virulence of various amoebic parasites has been correlated with the presence of electron-dense granules ( EDGs) in the cytoplasm of trophozoites. Here, we report the finding by transmission electron microscopy of a large number of EDGs in a recent culture of Acanthamoeba culbertsoni, isolated from a severe case of human keratitis. When this isolate was maintained in culture for 6 mo, the granules almost disappeared. However, after induction of mice brain lesions with the long-term cultured isolate, recovered amoebas had abundant EDGs. Trophozoites of the original isolate, or those recovered from experimental lesions, secreted EDGs into the medium when incubated with MDCK cells. To analyze a possible cytotoxic effect the conditioned medium was incubated with MDCK monolayers. After 5 h, the media containing EDGs produced opening of the tight junctions; at 24 h, cell viability was compromised, and at 48 h most of the cells were detached from the monolayer. In contrast, trophozoites in long-term cultures did not release EDGs to the medium during incubation with MDCK cells, and the corresponding conditioned medium did not have any effect on MDCK monolayers. Our observations further support the hypothesis that EDGs play a role in the cytopathogenic mechanisms of A. culbertsoni. [ABSTRACT FROM AUTHOR]

Acanthamoeba spp. are ubiquitous free‐living amoebae and do not need a host. They can, however, cause disease upon accidental contact with a potential host. The most common Acanthamoeba infection in humans is the so‐called Acanthamoeba keratitis (AK), occurring mainly in contact lens wearers. The first case of AK was reported in the early seventies and in the mid‐eighties the association between AK and contact lens wear was discovered. Today, acanthamoebae are among the most important causative agents of keratitis in contact lens wearers. AK often shows a severe progression, which is due to a lack of awareness but also to the lack of specific treatment. The dormant cysts pose a particular problem, often residing within the tissue and leading to reinfection after termination of treatment. In industrialized countries the annual incidence of AK lies between 0.1–1 cases per 100 000 inhabitants, with a marked regional variation depending on contact lens wear habits and mode of water supply. Our institution is the Austrian reference laboratory for Acanthamoeba diagnostics. All samples from patients with suspected AK are screened for Acanthamoeba spp. by culture and PCR in parallel, and all detected amoebae are genotyped. We currently have an annual incidence of AK of around 0.2 cases per 100 000 inhabitants, with >90% of cases occurring in contact lens wearers. In contrast to other countries, we do not see any seasonality and in contrast to the early years, today, males and females are equally represented, the most affected group being the 21–30‐year‐olds. The vast majority of cases are unilateral and many cases have a prolonged progression, in some cases infections remain viable for >1 yr. The predominant genotype is Acanthamoeba T4, other common genotypes are T3, T5, T6, T10 and T11. [ABSTRACT FROM AUTHOR]

The aim of this study was to identify and characterize the receptor system involved in controlling encystment in Acanthamoeba using specific agonists and antagonists and to examine whether endogenous stores of catecholamines are produced by the organism. Acanthamoeba trophozoites suspended in axenic growth medium were exposed to adrenoceptor agonists and antagonists to determine which compounds promoted or prevented encystment. Second, trophozoites were cultured in medium containing a catecholamine synthesis inhibitor to investigate the effect this had on natural encystment. Nonspecific adrenoceptor agonists including epinephrine, isoprotenerol, and the selective β1 adrenoceptor agonist dobutamine were found to cause > 90% encystment of Acanthamoeba trophozoites compared to < 30% with the controls. The selective β1 antagonist metoprolol was able to inhibit epinephrine mediated encystment by > 55%. Cultures of Acanthamoeba with the catecholamine synthesis inhibitor α-methyl-p-tyrosine significantly reduced the level of amoebic encystment compared to controls. In conclusion, Acanthamoeba appear to contain a functional adrenergic receptor system of unknown structure which is involved in initiating the encystment process that can be activated and blocked by β1 agonists and antagonists respectively. Furthermore, the presence of this receptor system in Acanthamoeba indicates that topical β adrenoceptor blockers may be effective adjunct therapy by reducing the transformation of trophozoites into the highly resistant cyst stage. [ABSTRACT FROM AUTHOR]

The case study targeted to determine the aetiology of nodular gill disease (NGD) of farmed rainbow trout. The methods included microscopical examination of gill material in fresh, culturing of isolated organisms, histology, transmission electron microscopy and molecular biology identification. The results revealed an intravital colonization of fish gills by the testate amoeba Rhogostoma minus Belar, 1921. Rhogostoma infection was found in all fish examined microscopically (15/15); in contrast, naked amoebae related to fully developed NGD lesions were found in minority of these fish (5/15). They belonged to four genera, Acanthamoeba, Vermamoeba, Naegleria and Vannella. Results presented in this study contribute to the mosaic of findings that contrary to amoebic gill disease of marine fish turn attention to the possibility of the heterogeneous, multi-amoeba-species and multifactorial aetiology of NGD. [ABSTRACT FROM AUTHOR]

Background Experimental studies have shown that the standard dose of riboflavin (R) or R + ultraviolet-A (UVA) as solo treatment are not able to exterminate Acanthamoeba cysts or even trophozoites. The purpose of this study is to determine whether the application of R + UVA can enhance the cysticidal effects of cationic antiseptic agents in vitro. Methods The log of either polyhexamethylene biguanide or chlorhexidine minimal cysticidal concentration in solutions containing riboflavin (concentrations 0.1, 0.05 and 0.025%) plus either Acanthamoeba castellanii cysts or Acanthamoeba polyphaga cysts was determined and compared in groups treated with UVA 30 mW/cm2 for 30 min and in control groups (with no exposure to UVA). A permutation test was used to determine the P value associated with treatment. Results Regardless of the riboflavin concentration and UVA treatment condition, no trophozoites were seen in plates where the cysts were previously exposed to cationic antiseptic agent concentrations ≥200 µg/mL for Acanthamoeba castellanii samples and ≥100 µg/mL for A. polyphaga samples. There was no statistical evidence that R + UVA treatment was associated with minimal cysticidal concentration ( P = 0.82). Conclusion R + UVA in doses up to 10 times higher than recommended for corneal crosslinking does not enhance the cysticidal effect of either polyhexamethylene biguanide or chlorhexidine in vitro. [ABSTRACT FROM AUTHOR]

Purpose Acanthamoeba keratitis is a severe, often sight threatening, corneal infection which in Western countries is predominantly seen in daily wear of contact lenses. This review aims to summarise the pathobiology and epidemiology of contact lens-related Acanthamoeba keratitis, and to present strategies for prevention, particularly with respect to modifiable risk factors in contact lens wear. Recent Findings The virulence of Acanthamoeba and resistance to treatment in keratitis appears to be linked with the production of a low molecular weight protease MIP133 by the organism, in response to binding to corneal epithelial cells through a mannose binding protein, and to the ability of the organism to convert from the trophozoite to the resistant cyst form. Recent epidemiological studies in contact lens relate disease have confirmed the link between solution topping up and Acanthamoeba keratitis and have reinforced the importance of avoidance of tap water, either as part of the care for the contact lens or storage case, handling lenses with wet hands or showering while wearing lenses. In the most recent analysis from the USA, there were no strong effects for solution type, water source or water disinfection process. Wearer age, lens wear time and history to appear to be linked with Acanthamoeba keratitis. Daily disposable contact lens use would be expected to reduce the prevalence of Acanthamoeba disease although this is unproven. Summary While Acanthamoeba keratitis remains challenging to diagnose and manage, strategies to limit the disease severity in contact lens wearers should include attention to recently identified risk factors, particularly those related to water contact. Public health awareness measures, the use of daily disposable contact lenses, a better understanding of the contribution of the host immunity and the development of standardised methods for culture of amoeba and testing of contact lens care systems against Acanthamoeba in the licensing process may be of value. Alternative treatments for the future may include those which target the mannose binding protein or the genes which control conversion to the cyst form. [ABSTRACT FROM AUTHOR]

ABSTRACT Acanthamoeba genus is divided into 20 genotypes (T1-T20) on the basis of the gene encoding 18S rRNA sequence. Using of at least 2 kbp gene fragments is strongly recommended to identify new genotypes and 5% difference is commonly used as a criterion of new genotypes, however, this value is questionable. In this paper, Polish Acanthamoeba strains described earlier on the basis of ~850 bp Ami fragment of 18S rRNA gene as T4, T11 and a new T16 genotype, have been analyzed using near-complete sequence of the gene. This analysis was needed because the Ami fragment does not reveal full variability within 18S rRNA gene. Phylogenetic analysis based on Ami fragment is biased by artifacts in the construction of the tree, so the fragment should not be used for identification of new putative Acanthamoeba genotypes. The analysis confirmed that the Polish sequences represent T4 and T11 genotypes and that the strains described earlier as T16 genotype are in fact a new subgroup of the T20 genotype and that this genotype should be divided into two subgroups: T20a (two strains described by [ J. Eukaryot. Microbiol. 62 (2015) 69]) and T20b (11 Polish strains described in this study). The T20b subgroup was isolated from both clinical samples and water bodies used by people as bathing places and there is a risk of infection for humans during contact with water. [ABSTRACT FROM AUTHOR]

Background: Helicobacter pylori (H. pylori) are resistant to hostile gastric environments and antibiotic therapy, reflecting the possibility that they are protected by an ecological niche, such as inside the vacuoles of human epithelial and immune cells. Acanthamoeba castellanii may also provide such an alternative niche, as fluorescently labeled H. pylori were observed as fast-moving and viable bacterium-like bodies inside the vacuoles of gastric, oral-fecal and water--food-borne Acanthamoeba castellanii. In addition, H. pylori-specific genes and proteins were detected in samples extracted from these ameba cells. The coccoid forms of H. pylori present within this ameba are converted to spiral form and produce peroxiredoxin and thiol peroxidase, providing the ability to detoxify oxygen metabolites formed in immune cells. Furthermore, these bacteria produce urease and vacA, two virulence determinants of H. pylori that influence phago-lysosome fusion and bacterial survival in macrophages. Microscopic observations of H. pylori cells in new generations of ameba along with amplification of H. pylori-specific genes from consecutive generations indicate that new ameba can inherit the intracellular H. pylori as part of their vacuolar content. Accordingly, it is proposed that Acanthamoeba vacuoles serve as sophisticated niche that protects H. pylori against the environmental stresses and provides essential nutrients for its growth and multiplication. This intracellular establishment inside the Acanthamoeba vacuole likely occurred long ago, leading to the adaptation of H. pylori to persist in phagocytic cells. The presence of these bacteria within Acanthamoeba, along with the transmission of Acanthamoeba from sewage, saliva water to others, provides explanations for the persistence and propagation of H. pylori in the human population. Objective: This topic highlights the reviews and discusses recent evidence regarding the evolutionary adaptation of H. pylori to thrive in host cell vacuoles. Method and Results: Helicobacter pylori (H. pylori) have been observed within Acanthamoeba vacuoles by light and fluorescence microscopy, and their presence has been confirmed by the detection of H. pylori-specific genes and proteins in Acanthamoeba extracts, such as vacA subunits, ureA, peroxiredoxin, and thiol peroxidase. Moreover, nonculturable H. pylori cells have been found in subsequent generations of Acanthamoeba, indicating the generational transmission of the bacteria as part of the transfer of vacuolar content. Conclusion: H.pylori are therefore well equipped to establish in the vacuoles of Acanthamoeba, which provide them with essential nutrients for multiplication, as a pre-adaptation for invasion of human cells. Due to general tropism of all bacteria to yeast extract, entrance of H. pylori into candida was modified by our experiments and it is corrected to "Acanthamoeba". [ABSTRACT FROM AUTHOR]

Free-living amoebae are protozoa widely distributed in nature, which can be found in a variety of environments. Four genera are recognized as causal agents of infections in humans and animals: Acanthamoeba, Naegleria, Balamuthia, and Sappinia. In this study, the presence of Acanthamoeba in dental units was determined and the isolates obtained were molecularly characterized; osmotolerance and thermotolerance assays were also performed to evaluate multiplication under these conditions, frequently associated with pathogenicity. The morphological analysis and partial sequencing of the 18S rDNA gene revealed the presence of Acanthamoeba genotype T4 in 14% of the units sampled. Osmotolerance and thermotolerance tests were positive for more than 80% of the isolates. Up to date, this is the first study that reports the detection, identification, and genotyping of Acanthamoeba isolated from dental units in Costa Rica and even in Latin-America. Further assays to determine the potential pathogenicity of these Acanthamoeba isolates are underway. [ABSTRACT FROM AUTHOR]

The 13th International Workshops on Opportunistic Protists ( IWOP-13) was held November 13-15, 2014 in Seville, Spain. The objectives of the IWOP meetings are to: (1) serve as a forum for exchange of new information among active researchers concerning the basic biology, molecular genetics, immunology, biochemistry, pathogenesis, drug development, therapy, and epidemiology of these immunodeficiency-associated pathogenic eukaryotic microorganisms that are seen in patients with AIDS and; (2) to foster the entry of new and young investigators into these underserved research areas. The IWOP meeting focuses on opportunistic protists; e.g. the free-living amoebae, Pneumocystis, Cryptosporidium, Toxoplasma, the Microsporidia, and kinetoplastid flagellates. This conference represents the major conference which brings together research groups working on these opportunistic pathogens. Progress has been achieved on understanding the biology of these pathogenic organisms, their involvement in disease causation in both immune deficient and immune competent hosts and is providing important insights into these emerging and reemerging pathogens. A continuing concern of the participants is the ongoing loss of scientific expertise and diversity in this research community. This decline is due to the small size of these research communities and an ongoing lack of understanding by the broader scientific community of the challenges and limitations faced by researchers working on these organisms, which makes these research communities very sensitive to declines in research funding. [ABSTRACT FROM AUTHOR]

Free-living amoebae are ubiquitous protozoa commonly found in water. Among them, Acanthamoeba and Vermamoeba (formerly Hartmannella) are the most represented genera. In case of stress, such as nutrient deprivation or osmotic stress, these amoebae initiate a differentiation process, named encystment. It leads to the cyst form, which is a resistant form enabling amoebae to survive in harsh conditions and resist disinfection treatments. Encystment has been thoroughly described in Acanthamoeba but poorly in Vermamoeba. Our study was aimed to follow the encystment/excystment processes by microscopic observations. We show that encystment is quite rapid, as mature cysts were obtained in 9 h, and that cyst wall is composed of two layers. A video shows that a locomotive form is likely involved in clustering cysts together during encystment. As for Acanthamoeba, autophagy is likely active during this process. Specific vesicles, possibly involved in ribophagy, were observed within the cytoplasm. Remarkably, mitochondria rearranged around the nucleus within the cyst, suggesting high needs in energy. Unlike Acanthamoeba and Naegleria, no ostioles were observed in the cyst wall suggesting that excystment is original. During excystment, large vesicles, likely filled with hydrolases, were found in close proximity to cyst wall and digest it. Trophozoite moves inside its cyst wall before exiting during excystment. In conclusion, Vermamoeba encystment/excystment displays original trends as compare to Acanthamoeba. [ABSTRACT FROM AUTHOR]

Acanthamoeba spp. are opportunistic pathogens that are ubiquitous in nature. Many species of this genus are responsible for a fatal encephalitis and keratitis in humans and other animals. Seventy-two soil samples were collected from the parishes across Jamaica and assessed for the presence of Acanthamoeba spp. Cultivation was carried out on non-nutrient agar plates seeded with heat killed Escherichia coli. PCR and sequencing of the DF3 region were carried out in order to genotype the isolated strains of Acanthamoeba. Thermotolerance and osmotolerance assays were utilized to investigate the pathogenic potential of the Acanthamoeba isolates. Acanthamoeba spp. was isolated from 63.9% of soil samples. Sequencing of the DF3 region of the 18S rDNA resulted in the identification of genotypes T4, T5, and T11. T4 genotype was most frequently isolated. Most isolates were thermotolerant or both thermotolerant and osmotolerant, indicating that they may present the potential to cause disease in humans and other animals. [ABSTRACT FROM AUTHOR]

Species of Acanthamoeba were first described using morphological characters including cyst structure and cytology of nuclear division. More than 20 nominal species were proposed using these methods. Morphology, especially cyst shape and size, has proven to be plastic and dependent upon culture conditions. The DNA sequence of the nuclear small-subunit (18S) rRNA, the Rns gene, has become the most widely accepted method for rapid diagnosis and classification of Acanthamoeba. The Byers-Fuerst lab first proposed an Rns typing system in 1996. Subsequent refinements, with an increasing DNA database and analysis of diagnostic fragments within the gene, have become widely accepted by the Acanthamoeba research community. The development of the typing system, including its current state of implementation is illustrated by three cases: (i) the division between sequence types T13 and T16; (ii) the diversity within sequence supertype T2/T6, and (iii) verification of a new sequence type, designated T20. Molecular studies make clear the disconnection between phylogenetic relatedness and species names, as applied for the genus Acanthamoeba. Future reconciliation of genetic types with species names must become a priority, but the possible shortcomings of the use of a single gene when reconstructing the evolutionary history of the acanthamoebidae must also be resolved. [ABSTRACT FROM AUTHOR]