Acanthamoeba keratitis: a review of biology, pathophysiology and epidemiology.

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.

Experimental keratitis in rats caused by Acanthamoeba griffini: A kinetic histopathological study.

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-1ß, 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.

In vitro effects of environmental isolates of Acanthamoeba T4 and T5 over human erythrocytes and platelets.

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

Acanthamoeba species isolated from Philippine freshwater systems: epidemiological and molecular aspects.

Free-living amoeba (FLA) research in the Philippines is still in its infancy but has, by far, demonstrated the presence of potentially pathogenic species. Acanthamoeba may cause sight-threatening and central nervous system infections to humans, yet its epidemiologic distribution from local environmental sources is yet to be defined. The present study aimed to provide a baseline epidemiologic distribution of Acanthamoeba spp. in freshwater systems in the Philippines and establish potential pathogenicity of isolates through thermo-tolerance assay. A total of 63 water samples were collected from 13 freshwater systems all over the Philippine archipelago. The low-volume (50 ml) water samples were processed and cultured on non-nutrient agar lawned with Escherichia coli and observed for amoebic growth using light microscopy. Amoebic culture demonstrated 14.28% (9/63) positivity while further molecular testing of culture-positive plates using Acanthamoeba-specific primers demonstrated 100% (9/9) confirmation of Acanthamoeba species. Genotyping of Acanthamoeba isolates revealed T1, T3, T4, T5, T7, T11, and T15 genotypes. Thermo-tolerance assay demonstrated that T5 and T7 genotypes were potentially pathogenic strains. The evidence of environmental distribution of Acanthamoeba spp. in the freshwater systems in the Philippines and thermo-tolerance profile of isolates are significant aspects of amoeba study in public health and calls for initiatives in the dissemination of relevant information and the expansion of knowledge, awareness, and policies on pathogenic waterborne amoeba to mitigate, prevent, detect, and report cases of human infections.

Molecular characterization of bacterial, viral and fungal endosymbionts of Acanthamoeba isolates in keratitis patients of Iran.

Free-living amoebae belong to the genus Acanthamoeba; can feed on microbial population by phagocytosis, and with the capability to act as a reservoir and a vehicle of microorganisms to susceptible host. Therefore, the role of endosymbiosis in the pathogenesis of Acanthamoeba is complex and not fully understood. The aim of the present study was to identify bacterial, fungal, and human adenovirus (HADV) endosymbionts as well as evaluating the endosymbionts role of such organisms in the pathogenesis of Acanthamoeba in keratitis patients living in Iran. Fifteen Acanthamoeba (T4 genotype) isolates were recovered from corneal scrapes and contact lenses of patients with keratitis. Cloning and purification was performed for all isolate. Gram staining was performed to identify bacterial endosymbionts. DNA extraction, PCR, and nested PCR was set up to identify endosymbiont of amoeba. Evaluation of pathogenicity was conducted by osmo-tolerance and thermo-tolerance assays and cell culture, and then CPE (cytopathic effect) was survey. Statistical analysis was used between Acanthamoeba associated endosymbionts and Acanthamoeba without endosymbiont at 24, 48, 72, and 96 h. A p value < 0.05 was considered as significant, statistically. A total of 9 (60%) Acanthamoeba (T4 genotypes) isolates were successfully cloned for detecting microorganism endosymbionts. The only isolate negative for the presence of endosymbiont was ICS9. ICS7 (Pseudomonas aeruginosa, Aspergillus sp., and human adenovirus endosymbionts) and ICS2 (Escherichia coli endosymbiont) isolates were considered as Acanthamoeba associated endosymbionts. ICS7 and ICS2 isolates were highly pathogen whereas ICS9 isolate showed low pathogenicity in pathogenicity evaluated. Positive CPE for ICS7 and ICS2 isolates and negative CPE for ICS9 isolate were observed in cell culture. The average number of cells, trophozoites, and cysts among ICS7, ICS2, and ICS9 isolates at 24, 48, 72, and 96 h was significant. This is the first survey on microbial endosymbionts of Acanthamoeba in keratitis patients of Iran, and also the first report of Aspergillus sp, Achromobacter sp., Microbacterium sp., Brevibacillus sp, Brevundimonas sp and Mastadenovirus sp in Acanthamoeba as endosymbionts. Our study demonstrated that microbial endosymbionts can affect the pathogenicity of Acanthamoeba; however, further research is required to clarify the exact pattern of symbiosis, in order to modify treatment protocol.

The modulatory effect of Artemisia annua L. on toll-like receptor expression in Acanthamoeba infected mouse lungs.

The genus Acanthamoeba, which may cause different infections in humans, occurs widely in the environment. Lung inflammation caused by these parasites induces pulmonary pathological changes such as pulmonary necrosis, peribronchial plasma cell infiltration, moderate desquamation of alveolar cells and partial destruction of bronchial epithelial cells, and presence of numerous trophozoites and cysts among inflammatory cells. The aim of this study was to assess the influence of plant extracts from Artemisia annua L. on expression of the toll-like receptors TLR2 and TLR4 in lungs of mice with acanthamoebiasis. A. annua, which belongs to the family Asteraceae, is an annual plant that grows wild in Asia. In this study, statistically significant changes of expression of TLR2 and TLR4 were demonstrated. In the lungs of infected mice after application of extract from A. annua the expression of TLRs was observed mainly in bronchial epithelial cells, pneumocytes (to a lesser extent during the outbreak of infection), and in the course of high general TLR expression. TLR4 in particular was also visible in stromal cells of lung parenchyma. In conclusion, we confirmed that a plant extract of A. annua has a modulatory effect on components of the immune system such as TLR2 and TLR4.

Pathogenic free-living amoebic encephalitis in Japan.

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.

Efficient therapeutic effect of Nigella sativa aqueous extract and chitosan nanoparticles against experimentally induced Acanthamoeba keratitis.

Acanthamoeba keratitis (AK) is a devastating, painful corneal infection, which may lead to loss of vision. The development of resistance and failure of the currently used drugs represent a therapeutic predicament. Thus, novel therapies with lethal effects on resistant Acanthamoeba are necessary to combat AK. In the present study, the curative effect of Nigella sativa aqueous extract (N. sativa) and chitosan nanoparticles (nCs) and both agents combined were assessed in experimentally induced AK. All inoculated corneas developed varying grades of AK. The study medications were applied on the 5th day postinoculation and were evaluated by clinical examination of the cornea and cultivation of corneal scraps. On the 10th day posttreatment, a 100% cure of AK was obtained with nCs (100 µg/ml) in grades 1 and 2 of corneal opacity as well as with N. sativa 60 mg/ml-nCs 100 µg/ml in grades 1, 2, and 3 of corneal opacity, highlighting a possible synergistic effect. On the 15th day posttreatment, a 100% cure was reached with N. sativa aqueous extract (60 mg/ml). Moreover, on the 20th day posttreatment, N. sativa (30 mg/ml) provided a cure rate of 87.5%, while nCs (50 µg/ml) as well as N. sativa 30 mg/ml-nCs 50 µg/ml yielded a cure rate of 75%; the lowest percentage of cure (25%) was obtained with chlorhexidine (0.02%), showing a non-significant difference compared to the parasite control. The clinical outcomes were in agreement with the results of corneal scrap cultivation. The results of the present study demonstrate the effectiveness of N. sativa aqueous extract and nCs (singly or combined) when used against AK, and these agents show potential for the development of new, effective, and safe therapeutic alternatives.

Acanthamoeba in Southeast Asia - Overview and Challenges.

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

Intra-amoebal killing of Mycobacterium ulcerans by Acanthamoeba griffini: A co-culture model.

Mycobacterium ulcerans, a decaying Mycobacterium marinum derivative is responsible for Buruli ulcer, a notifiable non-contagious disabling infection highly prevalent in some West African countries. Aquatic environments are suspected to host M. ulcerans, however, the exact reservoirs remain unknown. While M. marinum was found to resist amoebal microbicidal activities, this remains unknown for M. ulcerans. In this study M. ulcerans was co-cultured with the moderately halophile Acanthamoeba griffini at 30 °C to probe this tropical amoeba as a potential reservoir for M. ulcerans. In triplicate experiments, we observed engulfment of M. ulcerans by A. griffini trophozoites, followed by an unexpected significant difference of 98.4% (day 1), 99.5% (day 2), 99.5% (day 3) and 99.9% (day 7) between the number of intra-amoebal mycobacteria detected by PCR and the number of viable intra-amoebal mycobacteria measured by 10-week culture. Further encystment revealed only one Mycobacterium organism for 150 A. griffini cysts observed by electron microscopy and the culture of excysted amoebae remained sterile. In conclusion, these data install M. ulcerans as susceptible to A. griffini microbicidal activities rendering this amoeba species an unlikely host of M. ulcerans in natural environments.

Lyophilisation as a simple and safe method for long-term storage of free-living amoebae at ambient temperature.

Free-living amoebae (FLA) are protozoa ubiquitously found in nature. As some species or strains of these FLA are pathogenic for humans and animals, they represent objects of medical and parasitological research worldwide. Storage of valuable FLA strains in laboratories is often time- and energy-consuming and expensive. The shipment of such strains as frozen stocks is cumbersome and challenging in terms of cooling requirements as well as of transport regulations. To overcome these difficulties and challenges in maintenance and transport, we present a new method to generate lyophilised samples of non-cyst-forming FLA (Ripella (Vannella) spp.) and cyst-forming FLA (Acanthamoeba spp.) strains which guarantees a simple mechanism for long-term storage at ambient temperature, as well as easy handling and/or shipment. The survival rate of all FLA lyophilisates after short-term storage (2 months) was comparable to the survival rate of freeze cultures of the respective strains. Furthermore, the viability of Acanthamoeba spp. cysts after storage for 29 months was 20 to 40% following lyophilisation and rehydration, with strain variation.

Adhesion of Acanthamoeba on Cosmetic Contact Lenses.

BACKGROUND: This study aimed to evaluate the adhesion of Acanthamoeba trophozoites on cosmetic contact lenses (CLs) with and without CL care multipurpose solution (MPS) treatment. METHODS: Acanthamoeba lugdunensis L3a trophozoites were inoculated onto disks trimmed from CLs: 1-day Acuvue moist, 1-day Acuvue define, Acuvue 2, and Acuvue 2 define. After 18-hour inoculation, the number of adherent trophozoites was counted under phase contrast microscopy. The effects of MPS, Opti-Free Express, soaking CLs for 6 hours, on Acanthamoeba adhesion were analyzed. Scanning electron microscopic examination was performed for assessment of Acanthamoeba attached on the lens surface. RESULTS: Acanthamoeba trophozoites showed greater adhesion to cosmetic CL (P = 0.017 for 1-day CL and P = 0.009 for 2-week CL) although there was no significant difference between the types of cosmetic CL. On all lenses, the number of adherent Acanthamoeba was significantly reduced after treatment with MPS (P < 0.001 for 1-day Acuvue moist, P = 0.046 for 1-day Acuvue define, P < 0.001 for Acuvue 2, and P = 0.015 for Acuvue 2 define), but there was still significant difference between conventional and cosmetic CLs (P = 0.003 for 1-day CL and P < 0.001 for 2-week CL, respectively). More attachment of Acanthamoeba was observed on colored area and the acanthopodia of Acanthamoeba was placed on the rough surface of colored area. CONCLUSION: Acanthamoeba showed a greater affinity for cosmetic CL and mostly attached on colored area. Although MPS that contained myristamidopropyl dimethylamine reduced the adhesion rate, there was a significant difference between conventional and cosmetic CLs.

Expression and Activity of COX-1 and COX-2 in Acanthamoeba sp.-Infected Lungs According to the Host Immunological Status.

Little is known about the pathomechanism of pulmonary infections caused by Acanthamoeba sp. Therefore, the aim of this study was to determine whether Acanthamoeba sp. may affect the expression and activity of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), resulting in the altered levels of their main products, prostaglandins (PGE2) and thromboxane B2 (TXB2), in lungs of immunocompetent or immunosuppressed hosts. Acanthamoeba sp. induced a strong expression of COX-1 and COX-2 proteins in the lungs of immunocompetent mice, which, however, did not result in significant differences in the expression of PGE2 and TXB2. Our immunohistochemical analysis showed that immunosuppression induced by glucocorticoids in Acanthamoeba sp.-infected mice caused a decrease in COX-1 and COX-2 (not at the beginning of infection) in lung tissue. These results suggest that similar to COX-2, COX-1 is an important mediator of the pathophysiology in experimental pulmonary acanthamoebiasis. We suggest that the signaling pathways important for Acanthamoeba sp. induction of lung infection might interact with each other and depend on the host immune status.

Pathogenesis of microbial keratitis.

Microbial keratitis is a sight-threatening ocular infection caused by bacteria, fungi, and protist pathogens. Epithelial defects and injuries are key predisposing factors making the eye susceptible to corneal pathogens. Among bacterial pathogens, the most common agents responsible for keratitis include Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pneumonia and Serratia species. Fungal agents of corneal infections include both filamentous as well as yeast, including Fusarium, Aspergillus, Phaeohyphomycetes, Curvularia, Paecilomyces, Scedosporium and Candida species, while in protists, Acanthamoeba spp. are responsible for causing ocular disease. Clinical features include redness, pain, tearing, blur vision and inflammation but symptoms vary depending on the causative agent. The underlying molecular mechanisms associated with microbial pathogenesis include virulence factors as well as the host factors that aid in the progression of keratitis, resulting in damage to the ocular tissue. The treatment therefore should focus not only on the elimination of the culprit but also on the neutralization of virulence factors to minimize the damage, in addition to repairing the damaged tissue. A complete understanding of the pathogenesis of microbial keratitis will lead to the rational development of therapeutic interventions. This is a timely review of our current understanding of the advances made in this field in a comprehensible manner. Coupled with the recently available genome sequence information and high throughput genomics technology, and the availability of innovative approaches, this will stimulate interest in this field.

Resistance of Legionella and Acanthamoeba mauritaniensis to heat treatment as determined by relative and quantitative polymerase chain reactions.

Legionella and Acanthamoeba spp. persist in harvested rainwater pasteurized at high temperatures (> 72°C) and the interaction mechanisms exhibited between these organisms need to be elucidated. The resistance of two Legionella reference strains (Legionella pneumophila ATCC 33152 and Legionella longbeachae ATCC 33462), three environmental strains [Legionella longbeachae (env.), Legionella norrlandica (env.) and Legionella rowbothamii (env.)] and Acanthamoeba mauritaniensis ATCC 50676 to heat treatment (50-90°C) was determined by monitoring culturability and viability [ethidium monoazide quantitative polymerase chain reaction (EMA-qPCR)]. The expression of metabolic and virulence genes of L. pneumophila ATCC 33152 (lolA, sidF, csrA) and L. longbeachae (env.) (lolA) in co-culture with A. mauritaniensis ATCC 50676 during heat treatment (50-90°C) was monitored using relative qPCR. While the culturability (CFU/mL) and viability (gene copies/mL) of the Legionella strains reduced significantly (p < 0.05) following heat treatment (60-90°C), L. longbeachae (env.) and L. pneumophila ATCC 33152 were culturable following heat treatment at 50-60°C. Metabolically active trophozoites and dormant cysts of A. mauritaniensis ATCC 50676 were detected at 50°C and 60-90°C, respectively. For L. pneumophila ATCC 33152, lolA expression remained constant, sidF expression increased and the expression of csrA decreased during co-culture with A. mauritaniensis ATCC 50676. For L. longbeachae (env.), while lolA was up-regulated at 50-70°C, expression was not detected at 80-90°C and in co-culture. In conclusion, while heat treatment may reduce the number of viable Legionella spp. in monoculture, results indicate that the presence of A. mauritaniensis increases the virulence of L. pneumophila during heat treatment. The virulence of Legionella spp. in co-culture with Acanthamoeba spp. should thus be monitored in water distribution systems where temperature (heat) is utilized for treatment.

Interaction Between Methicillin-Resistant Staphylococcus aureus (MRSA) and Acanthamoeba polyphaga.

The interactions that occur between bacteria and amoebae can give through mutual relations, where both organisms benefit from the association or parasitic in which one organism benefits at the expense of the other. When these organisms share the same environment, it can result in some changes in the growth of organisms, in adaptation patterns, in morphology, development or even in their ability to synthesize proteins and other substances. In this study, the interaction between Acanthamoeba polyphaga and Staphylococcus aureus (MRSA) was evaluated using a co-culture model at different incubation times. The results showed that 89% of amoebic cells remained viable after contact with the bacteria. The bacterial isolate was visualized inside the amoeba through confocal microscopy and fluorescence for up to 216 h of co-cultivation. The lysate of amoebic culture increased the growth of S. aureus (MRSA), and the effect of supernatant of culture inhibited bacterial growth over the incubation times, suggesting that A. polyphaga produced some metabolite, that inhibited the growth of bacteria. Moreover, the encystment of the A. polyphaga was increased by the bacteria presence. The results show that A. polyphaga and S. aureus interaction may have an important influence on survival of both, and specially indicate a possible effect on the metabolics characteristics each other.

Acanthamoeba culbertsoni isolated from a clinical case with intraocular dissemination: Structure and in vitro analysis of the interaction with hamster cornea and MDCK epithelial cell monolayers.

Acanthamoeba culbertsoni trophozoites, previously isolated from a human keratitis case with severe intraocular damage, were maintained in axenic culture. Co-incubation of amoebae with MDCK cell monolayers demonstrated an apparent preference of the amoebae to introduce themselves between the cells. The trophozoites appeared to cross the cell monolayer through the tight junctions, which resulted in decreased trans-epithelial resistance (TER) measurements. Unexpectedly, after co-incubation of amoebae with hamster corneas, we observed that the trophozoites were able to cross the different cell layers and reach the corneal stroma after only 12 h of interaction, in contrast to other Acanthamoeba species. These observations suggest that this A. culbertsoni isolate is particularly pathogenic. Further research with diverse methodologies needs to be performed to explain the unique behavior of this Acanthamoeba strain.

Type 2 diabetes mellitus BALB/c mice are more susceptible to granulomatous amoebic encephalitis: Immunohistochemical study.

Granulomatous amoebic encephalitis (GAE) is a chronic, difficult to resolve infection caused by amphizoic amoebae of the genus Acanthamoeba, which in most cases occurs in immunosuppressed persons or with chronic diseases such as diabetes. In this study, we describe the early events of A. culbertsoni infection of GAE in diabetic mice model. Diabetes was induced in male BALB/c mice, with a dose of streptozotocin (130 mg/kg). Healthy and diabetic mice were inoculated via intranasal with 1 × 106 trophozoites of A. culbertsoni. Then were sacrificed and fixed by perfusion at 24, 48, 72 and 96 h post-inoculation, the brains and nasopharyngeal meatus were processed to immunohistochemical analysis. Invasion of trophozoites in diabetic mice was significantly greater with respect to inoculated healthy mice. Trophozoites and scarce cysts were immunolocalized in respiratory epithelial adjacent bone tissue, olfactory nerve packets, Schwann cells and the epineurium base since early 24 h post-inoculation. After 48 h, trophozoites were observed in the respiratory epithelium, white matter of the brain, subcortical central cortex and nasopharyngeal associated lymphoid tissue (NALT). At 72 h, cysts and trophozoites were immunolocalized in the olfactory bulb with the presence of a low inflammatory infiltrate characterized by polymorphonuclear cells. Scarce amoebae were observed in the granular layer of the cerebellum without evidence of inflammation or tissue damage. No amoebas were observed at 96 h after inoculation, suggesting penetration to other tissues at this time. In line with this, no inflammatory infiltrate was observed in the surrounding tissues where the amoebae were immunolocalized, which could contribute to the rapid spread of infection, particularly in diabetic mice. All data suggest that trophozoites invade the tissues by separating the superficial cells, penetrating between the junctions without causing cytolytic effect in the adjacent cells and subsequently reaching the CNS, importantly, diabetes increases the susceptibility to amoebae infection, which could favor the GAE development.

Acanthamoeba (T4) trophozoites cross the MDCK epithelium without cell damage but increase paracellular permeability and transepithelial resistance by modifying tight junction composition.

Free-living amoebae of the genus Acanthamoeba are protozoa ubiquitously found in nature. Some species of the genus are potentially pathogenic for humans provoking keratitis in healthy individuals, often in contact lens wearers and opportunistic infections such as pneumonitis, fatal granulomatous encephalitis and skin infections, particularly in immunocompromised individuals. The pathogenic mechanisms of these amoebae are poorly understood, however it had been suggested that contact dependent mechanisms are important during invasion, regardless of the epithelia type, since amoebae penetrate epithelia separating tight junction (TJ). This study was undertaken to determine whether Acanthamoeba sp. (T4) damages the barrier function of the TJ in MDCK epithelial monolayers. Actin cytoskeleton staining and electron microscopy analyses were performed; paracellular permeability and TJ sealing were evaluated by apicobasolateral diffusion of ruthenium red and transepithelial resistance (TER) measurements; immunofluorescence and Western blot assays were performed to locate and estimate expression of TJ protein claudins 2 (Cldn2) and 4 (Cldn4). The results show that Acanthamoeba sp. crosses the MDCK monolayer without altering the actin cytoskeleton or the morphology of the cells. When trophozoites or conditioned medium interact with the monolayer, paracellular diffusion of ruthenium red increases. After 6 h, the amoebae, but not their conditioned medium, increase the TER, and Cldn2 is removed from the TJ, and its overall content in the cells diminishes, while Cldn4 is targeted to the TJ without changing its expression level. In conclusion Acanthamoeba (T4) crosses MDCK monolayer without damaging the cells, increasing permeability and TER through Cldn2 degradation, and redirecting Cldn4 to TJ. These results strongly suggest that contact-dependent mechanisms are relevant during amoebae invasion.

Evaluation of the effectiveness of tea tree oil in treatment of Acanthamoeba infection.

Eye diseases caused by amoebae from the genus Acanthamoeba are usually chronic and severe, and their treatment is prolonged and not very effective. The difficulties associated with therapy have led to attempts at finding alternative treatment methods. Particularly popular is searching for cures among drugs made of plants. However, no substances with total efficacy in treating Acanthamoeba keratitis have been identified.Results of our semi in vivo studies of tea tree oil simulating eyeball infection demonstrated 100% effectiveness in the case of both trophozoites and cysts of amoebae from the genus Acanthamoeba. The action of tea tree oil indicates that this is the first substance with a potential ability to quickly and effectively remove the amoebae from the eye. Tea tree oil has the ability to penetrate tissues, which allows it to destroy amoebae in both the shallow and deep layers of the cornea. The present research into the use of tea tree oil in the therapy of Acanthamoeba infection is the first study of this type in parasitology. It offers tremendous potential for effective treatment of Acanthamoeba keratitis and other diseases caused by these protozoa.