Opportunistic free-living amoebal pathogens.

Pathogenic free-living amoebae affecting the central nervous system are known to cause granulomatous amoebic encephalitis (GAE) or primary amoebic meningoencephalitis (PAM). Although hosts with impaired immunity are generally at a higher risk of severe disease, amoebae such as Naegleria fowleri and Balamuthia mandrillaris can instigate disease in otherwise immunocompetent individuals, whereas Acanthamoeba species mostly infect immunocompromised people. Acanthamoeba also cause a sight-threatening eye infection, mostly in contact lens wearers. Although infections due to pathogenic amoebae are considered rare, recently, these deadly amoebae were detected in water supplies in the USA. This is of particular concern, especially with global warming further exacerbating the problem. Herein, we describe the epidemiology, presentation, diagnosis, and management of free-living amoeba infections.

Three encephalitis-causing amoebae and their distinct interactions with the host.

Naegleria fowleri, Balamuthia mandrillaris, and Acanthamoeba spp. can cause devastating brain infections in humans which almost always result in death. The symptoms of the three infections overlap, but brain inflammation and the course of the disease differ, depending on the amoeba that is responsible. Understanding the differences between these amoebae can result in the development of strategies to prevent and treat these infections. Recently, numerous scientific advancements have been made in the understanding of pathogenicity mechanisms in general, and the basic biology, epidemiology, and the human immune response towards these amoebae in particular. In this review, we combine this knowledge and aim to identify which factors can explain the differences between the lethal brain infections caused by N. fowleri, B. mandrillaris, and Acanthamoeba spp.

Studies on the cyst stage of Naegleria fowleri in vivo and in vitro.

Naegleria fowleri is a pathogenic, free-living amoeba that causes primary amebic meningoencephalitis (PAM), a highly fatal disease of the central nervous system. N. fowleri demonstrates three forms: the trophozoite, flagellate, and cyst. Most studies have focused on the trophozoite limiting information on the cyst. The present study examined the ability of cysts to attach to, excyst into the trophozoite form, and destroy cell cultures. Additionally, the study assessed the ability of cysts to cause PAM in a murine model. The results demonstrated that exposure to cysts and transformation into trophozoites resulted in destruction of cell cultures. Specifically, the mixed glial cells exhibited an increase in lactate dehydrogenase (LDH) release compared with cells without cyst exposure. On day eight postexposure, there was a nearly fourfold increase in LDH. The cysts of N. fowleri were shown not to be infective in vivo in a murine model. The mediation of the encystment process by the intracellular concentration of cAMP was also investigated. Trophozoites were treated with dipyridamole, an inhibitor of cAMP-specific phosphodiesterases. Dipyridamole increased the rate of encystment by nearly twofold and increased the intracellular concentration of cAMP in cysts by nearly sixfold throughout this period suggesting that cAMP is a mediator of encystment for N. fowleri.

Primary Amoebic Meningoencephalitis by Naegleria fowleri: Pathogenesis and Treatments.

Naegleria fowleri is a free-living amoeba (FLA) that is commonly known as the "brain-eating amoeba." This parasite can invade the central nervous system (CNS), causing an acute and fulminating infection known as primary amoebic meningoencephalitis (PAM). Even though PAM is characterized by low morbidity, it has shown a mortality rate of 98%, usually causing death in less than two weeks after the initial exposure. This review summarizes the most recent information about N. fowleri, its pathogenic molecular mechanisms, and the neuropathological processes implicated. Additionally, this review includes the main therapeutic strategies described in case reports and preclinical studies, including the possible use of immunomodulatory agents to decrease neurological damage.

Laurinterol from Laurencia johnstonii eliminates Naegleria fowleri triggering PCD by inhibition of ATPases.

Primary amoebic encephalitis (PAM) is a lethal disease caused by the opportunistic pathogen, Naegleria fowleri. This amoebic species is able to live freely in warm aquatic habitats and to infect children and young adults when they perform risk activities in these water bodies such as swimming or splashing. Besides the need to increase awareness of PAM which will allow an early diagnosis, the development of fully effective therapeutic agents is needed. Current treatment options are amphotericin B and miltefosine which are not fully effective and also present toxicity issues. In this study, the in vitro activity of various sesquiterpenes isolated from the red alga Laurencia johnstonii were tested against the trophozoite stage of a strain of Naegleria fowleri. Moreover, the induced effects (apoptotic cell death) of the most active compound, laurinterol (1), was evaluated by measuring DNA condensation, damages at the mitochondrial level, cell membrane disruption and production of reactive oxygen species (ROS). The obtained results demonstrated that laurinterol was able to eliminate the amoebae at concentrations of 13.42 ± 2.57 µM and also to induced programmed cell death (PCD) in the treated amoebae. Moreover, since ATP levels were highly affected and laurinterol has been previously reported as an inhibitor of the Na+/K+-ATPase sodium-potassium ion pump, comparison with known inhibitors of ATPases were carried out. Our results points out that laurinterol was able to inhibit ENA ATPase pump at concentrations 100 times lower than furosemide.

Copper detoxification machinery of the brain-eating amoeba Naegleria fowleri involves copper-translocating ATPase and the antioxidant system.

Copper is a trace metal that is necessary for all organisms but toxic when present in excess. Different mechanisms to avoid copper toxicity have been reported to date in pathogenic organisms such as Cryptococcus neoformans and Candida albicans. However, little if anything is known about pathogenic protozoans despite their importance in human and veterinary medicine. Naegleria fowleri is a free-living amoeba that occurs naturally in warm fresh water and can cause a rapid and deadly brain infection called primary amoebic meningoencephalitis (PAM). Here, we describe the mechanisms employed by N. fowleri to tolerate high copper concentrations, which include various strategies such as copper efflux mediated by a copper-translocating ATPase and upregulation of the expression of antioxidant enzymes and obscure hemerythrin-like and protoglobin-like proteins. The combination of different mechanisms efficiently protects the cell and ensures its high copper tolerance, which can be advantageous both in the natural environment and in the host. Nevertheless, we demonstrate that copper ionophores are potent antiamoebic agents; thus, copper metabolism may be considered a therapeutic target.

Primary Amebic Meningoencephalitis: A Case Report.

Primary amebic encephalitis (PAM) is a devastating central nervous system infection caused by Naegleria fowleri, a free-living amoeba, which can survive in soil and warm fresh water. Here, a 43-year-old healthy male was exposed to warm freshwater 5 days before the symptom onset. He rapidly developed severe cerebral edema before the diagnosis of PAM and was treated with intravenous conventional amphotericin B while died of terminal cerebral hernia finally. Comparing the patients with PAM who has similar clinical symptoms to those with other common types of meningoencephalitis, this infection is probably curable if treated early and aggressively. PAM should be considered in the differential diagnosis of purulent meningoencephalitis, especially in patients with recent freshwater-related activities during the hot season.

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.

The Effect of Different Environmental Conditions on the Viability of Naegleria fowleri Amoebae.

Naegleria fowleri, a free-living amoeba found in soil and freshwater environments, is the causative agent of Primary Amoebic Meningoencephalitis. Infection occurs when amoebae enter the nasal cavity, attach to the nasal mucosa and travel along olfactory neurons towards the olfactory bulb. Upon reaching the central nervous system, the amoebae replicate very rapidly and can cause death in 3-10 days. Little is known about the conditions in which the amoeba can survive in the environment. We have tested conditions beyond the known boundaries on the viability of amoebae by introducing them into moderate and extreme salinity, pH, and temperatures. Our data shows that although viability expectedly decreases towards each of these extreme conditions, their tolerance was much greater than anticipated, including viability in moderate salinity, a wide pH range, and temperatures higher than the previously reported 45 °C.

The therapeutic strategies against Naegleria fowleri.

Naegleria fowleri is a pathogenic amoeboflagellate most prominently known for its role as the etiological agent of the Primary Amoebic Meningoencephalitis (PAM), a disease that afflicts the central nervous system and is fatal in more than 95% of the reported cases. Although being fatal and with potential risks for an increase in the occurrence of the pathogen in populated areas, the organism receives little public health attention. A great underestimation in the number of PAM cases reported is assumed, taking into account the difficulty in obtaining an accurate diagnosis. In this review, we summarize different techniques and methods used in the identification of the protozoan in clinical and environmental samples. Since it remains unclear whether the protozoan infection can be successfully treated with the currently available drugs, we proceed to discuss the current PAM therapeutic strategies and its effectiveness. Finally, novel compounds for potential treatments are discussed as well as research on vaccine development against PAM.

Naegleria fowleri immunization modifies lymphocytes and APC of nasal mucosa.

We investigated whether intranasal immunization with amoebic lysates plus cholera toxin modified the populations of T and B lymphocytes, macrophages and dendritic cells by flow cytometry from nose-associated lymphoid tissue (NALT), cervical lymph nodes (CN), nasal passages (NP) and spleen (SP). In all immunized groups, the percentage of CD4 was higher than CD8 cells. CD45 was increased in B cells from mice immunized. We observed IgA antibody-forming cell (IgA-AFC) response, mainly in NALT and NP. Macrophages from NP and CN expressed the highest levels of CD80 and CD86 in N. fowleri lysates with either CT or CT alone immunized mice, whereas dendritic cells expressed high levels of CD80 and CD86 in all compartment from immunized mice. These were lower than those expressed by macrophages. Only in SP from CT-immunized mice, these costimulatory molecules were increased. These results suggest that N. fowleri and CT antigens are taking by APCs, and therefore, protective immunity depends on interactions between APCs and T cells from NP and CN. Consequently, CD4 cells stimulate the differentiation from B lymphocytes to AFC IgA-positive; antibody that we previously found interacting with trophozoites in the nasal lumen avoiding the N. fowleri attachment to nasal epithelium.

Brain-Eating Amoebae: Silver Nanoparticle Conjugation Enhanced Efficacy of Anti-Amoebic Drugs against Naegleria fowleri.

The overall aim of this study was to determine whether conjugation with silver nanoparticles enhances effects of available drugs against primary amoebic meningoencephalitis due to Naegleria fowleri. Amphotericin B, Nystatin, and Fluconazole were conjugated with silver nanoparticles, and synthesis was confirmed using UV-visible spectrophotometry. Atomic force microscopy determined their size in range of 20-100 nm. To determine amoebicidal effects, N. fowleri were incubated with drugs-conjugated silver nanoparticles, silver nanoparticles alone, and drugs alone. The findings revealed that silver nanoparticles conjugation significantly enhanced antiamoebic effects of Nystatin and Amphotericin B but not Fluconazole at micromolar concentrations, compared with the drugs alone. For the first time, our findings showed that silver nanoparticle conjugation enhances efficacy of antiamoebic drugs against N. fowleri. Given the rarity of the disease and challenges in developing new drugs, it is hoped that modifying existing drugs to enhance their antiamoebic effects is a useful avenue that holds promise in improving the treatment of brain-eating amoebae infection due to N. fowleri.

Efficient Liquid Media for Encystation of Pathogenic Free-Living Amoebae.

Pathogenic Naegleria fowleri, Acanthamoeba castellanii, and Acanthamoeba polyphaga, are distributed worldwide. They are causative agents of primary amoebic meningoencephalitis or acanthamoebic keratitis in humans, respectively. Trophozoites encyst in unfavorable environments, such as exhausted food supply and desiccation. Until recently, the method of N. fowleri encystation used solid non-nutrient agar medium supplemented with heat-inactivated Escherichia coli; however, for the amoebic encystment of Acanthamoeba spp., a defined, slightly modified liquid media is used. In this study, in order to generate pure N. fowleri cysts, a liquid encystment medium (buffer 1) modified from Page's amoeba saline was applied for encystation of N. fowleri. N. fowleri cysts were well induced after 24 hr with the above defined liquid encystment medium (buffer 1). This was confirmed by observation of a high expression of differential mRNA of nfa1 and actin genes in trophozoites. Thus, this liquid medium can replace the earlier non-nutrient agar medium for obtaining pure N. fowleri cysts. In addition, for cyst formation of Acanthamoeba spp., buffer 2 (adjusted to pH 9.0) was the more efficient medium. To summarize, these liquid encystment media may be useful for further studies which require axenic and pure amoebic cysts.

Effective PCR-based detection of Naegleria fowleri from cultured sample and PAM-developed mouse.

Increasing numbers of Primary Amoebic Meningoencephalitis (PAM) cases due to Naegleria fowleri are becoming a serious issue in subtropical and tropical countries as a Neglected Tropical Disease (NTD). To establish a rapid and effective diagnostic tool, a PCR-based detection technique was developed based on previous PCR methods. Four kinds of primer pairs, Nfa1, Nae3, Nf-ITS, and Naegl, were employed in the cultured amoebic trophozoites and a mouse with PAM experimentally developed by N. fowleri inoculation (PAM-mouse). For the extraction of genomic DNA from N. fowleri trophozoites (1×10(6)), simple boiling with 10µl of PBS (pH 7.4) at 100°C for 30min was found to be the most rapid and efficient procedure, allowing amplification of 2.5×10(2) trophozoites using the Nfa-1 primer. The primers Nfa1 and Nae3 amplified only N. fowleri DNA, whereas the ITS primer detected N. fowleri and N. gruberi DNA. Using the PAM-mouse brain tissue, the Nfa1 primer was able to amplify the N. fowleri DNA 4 days post infection with 1ng/µl of genomic DNA being detectable. Using the PAM-mouse CSF, amplification of the N. fowleri DNA with the Nae3 primer was possible 5 days post infection showing a better performance than the Nfa1 primer at day 6.

Development of a high- versus low-pathogenicity model of the free-living amoeba Naegleria fowleri.

Species in the genus Naegleria are free-living amoebae of the soil and warm fresh water. Although around 30 species have been recognized, Naegleria fowleri is the only one that causes primary amoebic meningoencephalitis (PAM) in humans. PAM is an acute and fast progressing disease affecting the central nervous system. Most of the patients die within 1-2 weeks of exposure to the infectious water source. The fact that N. fowleri causes such fast progressing and highly lethal infections has opened many questions regarding the relevant pathogenicity factors of the amoeba. In order to investigate the pathogenesis of N. fowleri under defined experimental conditions, we developed a novel high- versus low-pathogenicity model for this pathogen. We showed that the composition of the axenic growth media influenced growth behaviour and morphology, as well as in vitro cytotoxicity and in vivo pathogenicity of N. fowleri. Trophozoites maintained in Nelson's medium were highly pathogenic for mice, demonstrated rapid in vitro proliferation, characteristic expression of surface membrane vesicles and a small cell diameter, and killed target mouse fibroblasts by both contact-dependent and -independent destruction. In contrast, N. fowleri cultured in PYNFH medium exhibited a low pathogenicity, slower growth, increased cell size and contact-dependent target cell destruction. However, cultivation of the amoeba in PYNFH medium supplemented with liver hydrolysate (LH) resulted in trophozoites that were highly pathogenic in mice, and demonstrated an intermediate proliferation rate in vitro, diminished cell diameter and contact-dependent target cell destruction. Thus, in this model, the presence of LH resulted in increased proliferation of trophozoites in vitro and enhanced pathogenicity of N. fowleri in mice. However, neither in vitro cytotoxicity mechanisms nor the presence of membrane vesicles on the surface correlated with the pathologic potential of the amoeba. This indicated that the pathogenicity of N. fowleri remains a complex interaction between as-yet-unidentified cellular mechanisms.

Growth dynamic of Naegleria fowleri in a microbial freshwater biofilm.

The presence of pathogenic free-living amoebae (FLA) such as Naegleria fowleri in freshwater environments is a potential public health risk. Although its occurrence in various water sources has been well reported, its presence and associated factors in biofilm remain unknown. In this study, the density of N. fowleri in biofilms spontaneously growing on glass slides fed by raw freshwater were followed at 32 °C and 42 °C for 45 days. The biofilms were collected with their substrata and characterized for their structure, numbered for their bacterial density, thermophilic free-living amoebae, and pathogenic N. fowleri. The cell density of N. fowleri within the biofilms was significantly affected both by the temperature and the nutrient level (bacteria/amoeba ratio). At 32 °C, the density remained constantly low (1-10 N. fowleri/cm(2)) indicating that the amoebae were in a survival state, whereas at 42 °C the density reached 30-900 N. fowleri/cm(2) indicating an active growth phase. The nutrient level, as well, strongly affected the apparent specific growth rate (µ) of N. fowleri in the range of 0.03-0.23 h(-1). At 42 °C a hyperbolic relationship was found between µ and the bacteria/amoeba ratio. A ratio of 10(6) to 10(7) bacteria/amoeba was needed to approach the apparent µ(max) value (0.23 h(-1)). Data analysis also showed that a threshold for the nutrient level of close to 10(4) bacteria/amoeba is needed to detect the growth of N. fowleri in freshwater biofilm. This study emphasizes the important role of the temperature and bacteria as prey to promote not only the growth of N. fowleri, but also its survival.

Tap water or "sterile" water for sinus irrigations: what are our patients using?

BACKGROUND: Nasal saline irrigations are a valuable, widely used adjunct for the management of chronic rhinosinusitis. Due to potential concerns regarding infection, patients are commonly recommended to use distilled, bottled, or boiled tap water when mixing these solutions. Anecdotally, patients frequently inform otolaryngologists that they use tap water for irrigation preparation. The purpose of this study was to assess patient adherence to preparation guidelines. METHODS: This study was a cross-sectional, anonymous survey of 100 consecutive patients using nasal saline irrigations for chronic rhinosinusitis on the instruction of the senior author. Patients received their instructions in a standardized manner including printed handouts and had been instructed to use distilled, bottled, or boiled tap water. RESULTS: Patients almost uniformly reported improvement in their symptoms with the use of saline irrigations. No single water preparation was used by a majority of patients. However, tap water was used by 48% and the most common reason cited for using tap water was convenience. Of the patients using bottled, distilled, or boiled tap water, 65% described the process as "mildly" or "moderately" inconvenient. A large majority (70%) of patients report not adhering to cleaning instructions for their sinus rinse bottles. CONCLUSION: Despite standardized instructions for the preparation of saline irrigation solutions, many patients use untreated tap water. The extremely rare, but typically fatal, risk of meningoencephalitis from Naegleria fowlerii makes this a potential health hazard.