Modelling amoebic brain infection caused by Balamuthia mandrillaris using a human cerebral organoid.

The lack of disease models adequately resembling human tissue has hindered our understanding of amoebic brain infection. Three-dimensional structured organoids provide a microenvironment similar to human tissue. This study demonstrates the use of cerebral organoids to model a rare brain infection caused by the highly lethal amoeba Balamuthia mandrillaris. Cerebral organoids were generated from human pluripotent stem cells and infected with clinically isolated B. mandrillaris trophozoites. Histological examination showed amoebic invasion and neuron damage following coculture with the trophozoites. The transcript profile suggested an alteration in neuron growth and a proinflammatory response. The release of intracellular proteins specific to neuronal bodies and astrocytes was detected at higher levels postinfection. The amoebicidal effect of the repurposed drug nitroxoline was examined using the human cerebral organoids. Overall, the use of human cerebral organoids was important for understanding the mechanism of amoeba pathogenicity, identify biomarkers for brain injury, and in the testing of a potential amoebicidal drug in a context similar to the human brain.

Effectiveness of functional ingredients to enhance gill disease in Atlantic salmon (Salmo salar, L.).

The development and application of functional feed ingredients represents a great opportunity to advance fish growth and health, boost the immune system, and induce physiological benefits beyond those provided by traditional feeds. In the present study, we looked at the feasibility of in vitro methods for screening the qualities of functional feed ingredients using the fish cell line RTgill-W1, which has never been used in fish nutrition, and the culture of Paramoeba perurans. Five functional feed ingredients (arginine, ß-glucan, vitamin C, and two phytogenic feed additives) were selected to investigate their effects on cell viability and reactive oxygen species production. Three of the selected ingredients (arginine and two phytogenic feed additives) were additionally tested to assess their potential amoebicidal activity. As these functional ingredients are the core of a commercially available feed (Protec Gill, Skretting AS), their beneficial effects were further assessed in a field trial in fish affected by complex gill disease. Here, the analyzed parameters included the evaluation of macroscopic and histopathological gill conditions, pathogen detections, and analyses of plasma parameters. RTgill-W1 cell line assays were a good tool for screening functional ingredients and provided information about the optimal ingredient concentration ranges, which can be helpful for adjusting the concentrations in future feed diets. Through the culture of P. perurans, the tested ingredients showed a clear amoebicidal activity, suggesting that their inclusions in dietary supplements could be a viable way to prevent microbial infections. A three-week period of feeding Protec Gill slowed the disease progression, by reducing the pathogen load and significantly improving gill tissue conditions, as revealed by histological evaluation. The use of diets containing selected functional ingredients may be a feasible strategy for preventing or mitigating the increasingly common gill diseases, particularly in cases of complex gill disease, as documented in this study.

Metagenomic next-generation sequencing-assisted diagnosis of a rare case of primary cutaneous acanthamoebiasis in an HIV patient: a case report.

Pathogenic and free-living Acanthamoeba are widely distributed in the environment and have been reported to cause keratitis and universally fatal encephalitis. Primary cutaneous acanthamoebiasis caused by Acanthamoeba is exceedingly rare and presents as isolated necrotic cutaneous lesions without involvement of the cornea or central nervous system. Cutaneous acanthamoebiasis often occurs in immunocompromised patients and is likely overlooked or even misdiagnosed only by cutaneous biopsy tissue histopathological analysis. Here, we report a HIV-infected 63-year-old female with oral leukoplakia for 4 months and scattered large skin ulcers all over the body for 2 months. The cause of the cutaneous lesions was unclear through cutaneous specimens histopathological analysis, and subsequently Acanthamoeba were detected by metagenomic next-generation sequencing (mNGS), which may be the cause of cutaneous lesions. Based on the mNGS results, a pathologist subsequently reviewed the previous pathological slides and found trophozoites of Acanthamoeba so that the cause was identified, and the skin ulcers improved significantly after treatment with multi-drug combination therapy. Acanthamoeba is also a host of pathogenic microorganisms. The presence of endosymbionts enhances the pathogenicity of Acanthamoeba, and no other pathogens were reported in this case. mNGS is helpful for rapidly diagnosing the etiology of rare skin diseases and can indicate the presence or absence of commensal microorganisms.

Flucofuron as a Promising Therapeutic Agent against Brain-Eating Amoeba.

Primary amoebic meningoencephalitis (PAM) is a rare and fulminant neurodegenerative disease caused by the free-living amoeba Naegleria fowleri. Currently, there is a lack of standardized protocols for therapeutic action. In response to the critical need for effective therapeutic agents, we explored the Global Health Priority Box, a collection of 240 compounds provided by the Medicines for Malaria Venture (MMV). From this pool, flucofuron emerged as a promising candidate, exhibiting high efficacy against trophozoites of both N. fowleri strains (ATCC 30808 IC50 : 2.58 ± 0.64 µM and ATCC 30215 IC50: 2.47 ± 0.38 µM), being even active against the resistant cyst stage (IC50: 0.88 ± 0.07 µM). Moreover, flucofuron induced diverse metabolic events that suggest the triggering of apoptotic cell death. This study highlights the potential of repurposing medications for treating challenging diseases, such as PAM.

Isolation, characterization, and pathogenicity assay of Acanthamoeba and its endosymbionts in respiratory disorders and COVID-19 hospitalized patients, northern Iran.

Acanthamoeba spp., are common free-living amoebae found in nature that can serve as reservoirs for certain microorganisms. The SARS-CoV-2 virus is a newly emerged respiratory infection, and the investigation of parasitic infections remains an area of limited research. Given that Acanthamoeba can act as a host for various endosymbiotic microbial pathogens and its pathogenicity assay is not fully understood, this study aimed to identify Acanthamoeba and its bacterial and fungal endosymbionts in patients with chronic respiratory disorders and hospitalized COVID-19 patients in northern Iran. Additionally, a pathogenicity assay was conducted on Acanthamoeba isolates. Urine, nasopharyngeal swab, and respiratory specimens were collected from two groups, and each sample was cultured on 1.5% non-nutrient agar medium. The cultures were then incubated at room temperature and monitored daily for a period of two weeks. Eight Acanthamoeba isolates were identified, and PCR was performed to confirm the presence of amoebae and identify their endosymbionts. Four isolates were found to have bacterial endosymbionts, including Stenotrophomonas maltophilia and Achromobacter sp., while two isolates harbored fungal endosymbionts, including an uncultured fungus and Gloeotinia sp. In the pathogenicity assay, five isolates exhibited a higher degree of pathogenicity compared to the other three. This study provides significant insights into the comorbidity of acanthamoebiasis and COVID-19 on a global scale, and presents the first evidence of Gloeotinia sp. as a fungal endosymbiont. Nevertheless, further research is required to fully comprehend the symbiotic patterns and establish effective treatment protocols.

Identification of new amoebae strains in rainbow trout (Oncorhynchus mykiss, Walbaum) farms affected by nodular gill disease (NGD) in Northeastern Italy.

Nodular gill disease (NGD) is an emerging condition associated with amoeba trophozoites in freshwater salmonid farms. However, unambiguous identification of the pathogens still must be achieved. This study aimed to identify the amoeba species involved in periodic NGD outbreaks in two rainbow trout (Oncorhynchus mykiss) farms in Northeastern Italy. During four episodes (February-April 2023), 88 fish were euthanized, and their gills were evaluated by macroscopic, microscopic and histopathological examination. The macroscopic and microscopic severity of the lesions and the degree of amoebae infestation were scored and statistically evaluated. One gill arch from each animal was put on non-nutrient agar (NNA) Petri dishes for amoeba isolation, cultivation and subsequent identification with SSU rDNA sequencing. Histopathology confirmed moderate to severe lesions consistent with NGD and mild to moderate amoeba infestation. The presence of amoebae was significantly correlated with lesion severity. Light microscopy of cultured amoebae strains and SSU rDNA analysis revealed the presence of a previously characterized amoeba Naegleria sp. strain GERK and several new strains: two strains from Hartmannelidae, three vannelid amoebae from the genus Ripella and cercozoan amoeba Rosculus. Despite the uncertainty in NGD etiopathogenesis and amoebae pathogenic role, identifying known and new amoebae leans towards a possible multi-aetiological origin.

Phylogenetic analysis of Acanthamoeba isolated from soil samples and nasal cavities of patients with malignancy: a public health concern in the northwest of Iran.

BACKGROUND: The genus Acanthamoeba is reported from various environmental sources and can cause multiple complications, including chronic amoebic aeratitis and amoebic granulomatous encephalitis. This study investigated the presence and genotyping of Acanthamoeba in the soil of parks and patients with malignancies referred to health centers in Zanjan city, Iran. METHODS: In this cross-sectional study, 200 soil samples were collected from amusement parks in Zanjan city from September 2017 to May 2018. Samples were cultured on 1.5% non-nutrient agar, and the Acanthamoeba genus was identified using the morphological method. PCR was performed on all positive environmental samples, and six microscopically positive clinical samples belonged to our previous study. DNA sequencing of 18S rRNA was performed to analyze the genetic pattern of some PCR-positive isolates. RESULTS: Microscopic results showed that 96 (48%) soil samples were positive. PCR confirmed all positive cases of clinical samples and 84 soil samples. Out of the PCR-positive samples, 20 soil samples and five clinical samples were sequenced successfully. All soil isolates belonged to the T4 genotype, and three and two clinical samples belonged to T4 and T5 genotypes, respectively. CONCLUSION: : The presence of Acanthamoeba in both the environment and clinical samples of Zanjan city suggests paying greater attention to the infections caused by it.

First Report of Isolation and Characterization of Acanthamoeba spp. from the Milk Used for Calf Feeding.

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

The gene expression and proteomic profiling of Acanthamoeba isolates.

INTRODUCTION: The free-living protozoan Acanthamoeba can cause severe keratitis known as Acanthamoeba Keratitis (AK) and granulomatous amoebic encephalitis (GAE). The pathogenesis of Acanthamoeba includes intricate interactions between the organism and the host's immune system. The downstream analysis of a well-annotated genome assembly along with proteomic analysis can unravel several biological processes and aid in the identification of potential genes involved in pathogenicity. METHODS: Based on the next-generation sequencing data analysis, genes including lysophospholipase, phospholipase, S8/S53 peptidase, carboxylesterase, and mannose-binding protein were selected as probable pathogenic targets that were validated by conventional PCR in a total of 30 Acanthamoeba isolates. This was followed by real-time PCR for the evaluation of relative gene expression in the keratitis and amoebic encephalitis animal model induced using keratitis (CHA5), encephalitis (CHA24) and non-pathogenic environmental isolate (CHA36). In addition, liquid chromatography-mass spectrometry (LC-MS/MS) was performed for keratitis, encephalitis, and non-pathogenic environmental isolate before and after treatment with polyhexamethylene biguanide (PHMB). RESULTS: The conventional PCR demonstrated the successful amplification of lysophospholipase, phospholipase, S8/S53 peptidase, carboxylesterase, and mannose-binding protein genes in clinical and environmental isolates. The expression analysis revealed phospholipase, lysophospholipase, and mannose-binding genes to be significantly upregulated in the keratitis isolate (CHA 5) during AK in the animal model. In the case of the amoebic encephalitis model, phospholipase, lysophospholipase, S8/S53 peptidase, and carboxylesterase were significantly upregulated in the encephalitis isolate compared to the keratitis isolate. The proteomic data revealed differential protein expression in pathogenic versus non-pathogenic isolates in the pre and post-treatment with PHMB. CONCLUSION: The gene expression data suggests that lysophospholipase, phospholipase, S8/S53 peptidase, carboxylesterase, and mannose-binding protein (MBP) could play a role in the contact-dependent and independent mechanisms of Acanthamoeba pathogenesis. In addition, the proteomic profiling of the 3 isolates revealed differential protein expression crucial for parasite growth, survival, and virulence. Our results provide baseline data for selecting possible pathogenic targets that could be utilized for designing knockout experiments in the future.

Amoebiasis: Advances in Diagnosis, Treatment, Immunology Features and the Interaction with the Intestinal Ecosystem.

This review of human amoebiasis is based on the most current knowledge of pathogenesis, diagnosis, treatment, and Entamoeba/microbiota interactions. The most relevant findings during this last decade about the Entamoeba parasite and the disease are related to the possibility of culturing trophozoites of different isolates from infected individuals that allowed the characterization of the multiple pathogenic mechanisms of the parasite and the understanding of the host-parasite relationship in the human. Second, the considerable advances in molecular biology and genetics help us to analyze the genome of Entamoeba, their genetic diversity, and the association of specific genotypes with the different amoebic forms of human amoebiasis. Based on this knowledge, culture and/or molecular diagnostic strategies are now available to determine the Entamoeba species and genotype responsible for invasive intestinal or extraintestinal amoebiasis cases. Likewise, the extensive knowledge of the immune response in amoebiasis with the appearance of new technologies made it possible to design diagnostic tools now available worldwide. Finally, the understanding of the interaction between the Entamoeba species and the intestinal microbiota aids the understanding of the ecology of this parasite in the human environment. These relevant findings will be discussed in this review.

Assessment of pathogenic potential of Acanthamoeba isolates by in vitro and in vivo tests.

Acanthamoeba are free-living protozoa present ubiquitously in numerous environmental reservoirs that exist as an actively feeding trophozoite or a dormant cyst stage. The pathogenic Acanthamoeba are known to cause Acanthamoeba keratitis (AK) and granulomatous amoebic encephalitis (GAE). Despite their omnipresence, the number of infections is quite low. The reason behind this low frequency of Acanthamoeba infections could be the existence of many non-pathogenic strains or a successful host immune response to these infections. Studies in the past have proposed a few physiological parameters for the differentiation of pathogenic and non-pathogenic strains. Additionally, in vivo experiments are known to play an essential role in understanding the virulence of parasites, immunological aspects, and disease pathogenesis. The thermotolerance (30 °C, 37 °C, and 40 °C) and osmotolerance (0.5 M, 1 M, and 1.5 M) tests were performed on 43 Acanthamoeba isolates from patients with keratitis (n = 22), encephalitis (n = 5), and water samples (n = 16). In addition, the genotype of 10 Acanthamoeba isolates (keratitis (n = 2), encephalitis (n = 2), water (n = 6)) was determined and were then evaluated for pathogenicity on mouse model by inducing Acanthamoeba keratitis and amoebic encephalitis. The results of the thermotolerance and osmotolerance assays categorized 29/43 (67.4%) isolates as pathogenic, 8 as low pathogenic (18.6%), and the remaining 6 (13.9%) as non-pathogenic. The 10 Acanthamoeba isolates were categorized as T11 (5 isolates), T5 (2 isolates), T4 (2 isolates), and T10 (1 isolate) genotypes. Out of 10 Acanthamoeba isolates, 9 were successful in establishing AK, amoebic encephalitis, or both in the mice model, and a single isolate was found non-pathogenic. Two isolates from water samples were non-pathogenic in the physiological tests but successfully established Acanthamoeba infection in the mice model. The results of the physiological assays and in vivo experiments were analogous for 7 isolates while 1 isolate from the water was low pathogenic in the physiological assays but failed to produce pathogenicity during in vivo experiments. The physiological parameters are not very dependable to test the pathogenic potential of Acanthamoeba isolates, and thus results must always be validated by in vivo experiments. There is no infallible approach for determining the potential pathogenicity of environmental isolates of Acanthamoeba because several parameters regulate the pathogenic potential.

Anti-Balamuthia mandrillaris and anti-Naegleria fowleri effects of drugs conjugated with various nanostructures.

Balamuthia mandrillaris and Naegleria fowleri are protist pathogens that can cause fatal infections. Despite mortality rate of > 90%, there is no effective therapy. Treatment remains problematic involving repurposed drugs, e.g., azoles, amphotericin B and miltefosine but requires early diagnosis. In addition to drug discovery, modifying existing drugs using nanotechnology offers promise in the development of therapeutic interventions against these parasitic infections. Herein, various drugs conjugated with nanoparticles were developed and evaluated for their antiprotozoal activities. Characterizations of the drugs' formulations were accomplished utilizing Fourier-transform infrared spectroscopy, efficiency of drug entrapment, polydispersity index, zeta potential, size, and surface morphology. The nanoconjugates were tested against human cells to determine their toxicity in vitro. The majority of drug nanoconjugates exhibited amoebicidal effects against B. mandrillaris and N. fowleri. Amphotericin B-, Sulfamethoxazole-, Metronidazole-based nanoconjugates are of interest since they exhibited significant amoebicidal effects against both parasites (p < 0.05). Furthermore, Sulfamethoxazole and Naproxen significantly diminished host cell death caused by B. mandrillaris by up to 70% (p < 0.05), while Amphotericin B-, Sulfamethoxazole-, Metronidazole-based drug nanoconjugates showed the highest reduction in host cell death caused by N. fowleri by up to 80%. When tested alone, all of the drug nanoconjugates tested in this study showed limited toxic effects against human cells in vitro (less than 20%). Although these are promising findings, prospective work is warranted to comprehend the mechanistic details of nanoconjugates versus amoebae as well as their in vivo testing, to develop antimicrobials against the devastating infections caused by these parasites.

A clinical case report of Balamuthia granulomatous amoebic encephalitis in a non-immunocompromised patient and literature review.

BACKGROUND: Balamuthia granulomatous amoebic encephalitis (GAE) is a peculiar parasitic infectious disease of the central nervous system, about 39% of the infected Balamuthia GAE patients were found to be immunocompromised and is extremely rare clinically. The presence of trophozoites in diseased tissue is an important basis for pathological diagnosis of GAE. Balamuthia GAE is a rare and highly fatal infection for which there is no effective treatment plan in clinical practice. CASE PRESENTATION: This paper reports clinical data from a patient with Balamuthia GAE to improve physician understanding of the disease and diagnostic accuracy of imaging and reduce misdiagnosis. A 61-year-old male poultry farmer presented with moderate swelling pain in the right frontoparietal region without obvious inducement three weeks ago. Head computed tomography(CT) and magnetic resonance imaging(MRI) revealed a space-occupying lesion in the right frontal lobe. Intially clinical imaging diagnosed it as a high-grade astrocytoma. The pathological diagnosis of the lesion was inflammatory granulomatous lesions with extensive necrosis, suggesting amoeba infection. The pathogen detected by metagenomic next-generation sequencing (mNGS) is Balamuthia mandrillaris, the final pathological diagnosis was Balamuthia GAE. CONCLUSION: When a head MRI shows irregular or annular enhancement, clinicians should not blindly diagnose common diseases such as brain tumors. Although Balamuthia GAE accounts for only a small proportion of intracranial infections, it should be considered in the differential diagnosis.

Role of FcγRIII in the nasal cavity of BALB/c mice in the primary amebic meningoencephalitis protection model.

Different mechanisms of the host immune response against the primary amebic meningoencephalitis (PAM) in the mouse protection model have been described. It has been proposed that antibodies opsonize Naegleria fowleri trophozoites; subsequently, the polymorphonuclear cells (PMNs) surround the trophozoites to avoid the infection. FcγRs activate signaling pathways of adapter proteins such as Syk and Hck on PMNs to promote different effector cell functions which are induced by the Fc portion of the antibody-antigen complexes. In this work, we analyzed the activation of PMNs, epithelial cells, and nasal passage cells via the expression of Syk and Hck genes. Our results showed an increment of the FcγRIII and IgG subclasses in the nasal cavity from immunized mice as well as Syk and Hck expression was increased, whereas in the in vitro assay, we observed that when the trophozoites of N. fowleri were opsonized with IgG anti-N. fowleri and interacted with PMN, the expression of Syk and Hck was also increased. We suggest that PMNs are activated via their FcγRIII, which leads to the elimination of the trophozoites in vitro, while in the nasal cavity, the adhesion and consequently infection are avoided.

Cinnamic acid and lactobionic acid based nanoformulations as a potential antiamoebic therapeutics.

Acanthamoeba castellanii causes granulomatous amoebic encephalitis, an uncommon but severe brain infection and sight-threatening Acanthamoeba keratitis. Most of the currently used anti-amoebic treatments are not always effective, due to persistence of the cyst stage, and recurrence can occur. Here in this study we synthesize cinnamic acid and lactobionic acid-based magnetic nanoparticles (MNPs) using co-precipitation technique. These nanoformulations were characterized by Fourier transform infrared spectroscopy and Atomic form microscopy. The drugs alone (Hesperidin, Curcumin and Amphotericin B), magnetic NPs alone, and drug-loaded nano-formulations were evaluated at a concentration of 100 µg/mL for antiamoebic activity against a clinical isolate of A. castellanii. Amoebicidal assays revealed that drugs and conjugation of drugs and NPs further enhanced amoebicidal effects of drug-loaded nanoformulations. Drugs and drug-loaded nanoformulations inhibited both encystation and excystation of amoebae. In addition, drugs and drug-loaded nanoformulations inhibited parasite binding capability to the host cells. Neither drugs nor drug-loaded nanoformulations showed cytotoxic effects against host cells and considerably reduced parasite-mediated host cell death. Overall, these findings imply that conjugation of medically approved drugs with MNPs produce potent anti-Acanthamoebic effects, which could eventually lead to the development of therapeutic medications.

Pathogenicity and virulence of Entamoeba histolytica, the agent of amoebiasis.

The amoeba parasite Entamoeba histolytica is the causative agent of human amebiasis, an enteropathic disease affecting millions of people worldwide. This ancient protozoan is an elementary example of how parasites evolve with humans, e.g. taking advantage of multiple mechanisms to evade immune responses, interacting with microbiota for nutritional and protective needs, utilizing host resources for growth, division, and encystation. These skills of E. histolytica perpetuate the species and incidence of infection. However, in 10% of infected cases, the parasite turns into a pathogen; the host-parasite equilibrium is then disorganized, and the simple lifecycle based on two cell forms, trophozoites and cysts, becomes unbalanced. Trophozoites acquire a virulent phenotype which, when non-controlled, leads to intestinal invasion with the onset of amoebiasis symptoms. Virulent E. histolytica must cross mucus, epithelium, connective tissue and possibly blood. This highly mobile parasite faces various stresses and a powerful host immune response, with oxidative stress being a challenge for its survival. New emerging research avenues and omics technologies target gene regulation to determine human or parasitic factors activated upon infection, their role in virulence activation, and in pathogenesis; this research bears in mind that E. histolytica is a resident of the complex intestinal ecosystem. The goal is to eradicate amoebiasis from the planet, but the parasitic life of E. histolytica is ancient and complex and will likely continue to evolve with humans. Advances in these topics are summarized here.

Aislamiento de Acanthamoeba spp. en salas cerradas de un hospital de la provincia de Buenos Aires, Argentina / Isolation of Acanthamoeba spp. in closed rooms of a hospital in Buenos Aires province, Argentina

Resumen Las amebas de vida libre (AVL) son protozoos ubicuos con cuatro géneros patógenos para el ser humano: Acanthamoeba, Naegleria, Balamuthia, y Sappinia. Acanthamoeba puede actuar como reservorio de microorganismos (endosimbiontes), por lo cual, en medio hospitalario, implicaría un riesgo para la transmisión de bacterias, virus y hongos intranosocomiales. Se investigó la presencia de AVL, con énfasis en Acanthamoeba spp., en un hospital pediátrico de la provincia de Buenos Aires, Argentina. Se colectaron 22 muestras de lavamanos e incubadoras en salas de Neonatología y Terapia Intensiva, las que fueron cultivadas a 37 y 42 °C. Los aislados fueron identificados molecularmente. El 63,64% de las muestras presentaron Acanthamoeba spp. Esta investigación representa el primer estudio realizado en la Argentina sobre la detección de Acanthamoeba spp. en salas cerradas de un hospital. Su presencia es una señal de alarma y resulta un blanco útil para investigar posibles reservorios de microorganismos patógenos en ambientes hospitalarios.

Abstract Free-living amoebae (FLA) are ubiquitous protozoa with four pathogenic genera for humans: Acanthamoeba, Naegleria, Balamuthia, and Sappinia. Acanthamoeba can act as a reservoir of microorganisms (endosymbionts), for which reason, in a hospital environment, it would imply a risk for transmission of nosocomial bacteria, viruses and fungi. The presence of AVL, with emphasis on Acanthamoeba spp., was investigated in a pediatric hospital. Twenty-two samples were collected from sinks and incubators in Neonatology and Intensive Care rooms, which were cultured at 37 and 42 °C. The isolates found were molecularly identified. A total of 63.64% of the samples presented Acanthamoeba spp. This research represents the first study in Argentina on the detection of Acanthamoeba spp. in closed rooms of a hospital. Its presence is an alarm signal, and it is a useful target to investigate possible reservoirs of pathogenic microorganisms in hospital environments.

Resumo As amebas de vida livre (AVL) são protozoários ubíquos com quatro gêneros patogênicos para o ser humano: Acanthamoeba, Naegleria, Balamuthia, e Sappinia. Acanthamoeba pode atuar como um reservatório de microrganismos (endossimbiontes), e portanto, em um ambiente hospitalar, representaria um risco de transmissão de bactérias, vírus e fungos intra-nosocomiais. A presença de AVL, com em Acanthamoeba spp. em um hospital pediátrico da província de Buenos Aires, Argentina, foi investigada. Vinte e duas amostras foram coletadas em lavatórios e incubadoras em Salas de Neonatologia e Cuidados Intensivos, cultivadas a 37 e 42 °C. Os isolados foram identificadas molecularmente. Foram encontradas Acanthamoeba spp. em 63,64% das amostras. Esta investigação representa o primeiro estudo realizado na Argentina sobre a detecção de Acanthamoeba spp. em salas fechadas de um hospital. A sua presença é um sinal de alarme e um alvo para investigar possíveis reservatórios de microrganismos patogênicos em ambientes hospitalares.

Occurrence of Naegleria fowleri and their implication for health - a look under the One Health approaches.

One Health approaches are becoming increasingly necessary in the world we live in. Human beings, animals, plants and the environment are intrinsically interconnected and when some intervention occurs, mainly through the action of man himself, everyone suffers the consequences. The objective of this review was to collect data about the occurrence and dispersion of Naegleria fowleri, an amphizoic free-living amoeba, and its implications for health approaches through the One Health concept. N. fowleri is an opportunistic amoeba, better known as brain-eating amoeba, which causes Primary Amoebic Meningoencephalitis. This amoeba is widely distributed around the world, being isolated from different matrices of natural or anthropogenic environments with temperatures above 30 °C with an upper limit of 45-46 °C. Highly lethal, it has claimed numerous humans patients and only five people have survived the disease so far. Our results indicate that climate change plays a major role in the growth and dispersion of the pathogen in the environment, causing damage to humans and animals. Changes in temperature, antimicrobial resistance, possible transport of other microorganisms by the amoeba, conventional treatments with chlorination, among others, were addressed in our study and should be considered in order to raise questions and possible solutions to this problem that involves health as a whole. The diagnostic methods, prospection of new anti-Naegleria drugs and the control of this parasite in the environment are specific and urgent issues. We know that the human-animal-plants-environment spheres are inseparable, so it is necessary to turn a directed look at the One Health approaches related to N. fowleri.

Regulation of the molecular repertoires of oxidative stress response in the gills and olfactory organ of Atlantic salmon following infection and treatment of the parasite Neoparameoba perurans.

The present study investigated the involvement of key molecular regulators of oxidative stress in amoebic gill disease (AGD), a parasitic infestation in Atlantic salmon. In addition, the study evaluated how these molecular biomarkers responded when AGD-affected fish were exposed to a candidate chemotherapeutic peracetic acid (PAA). Atlantic salmon were experimentally infected with the parasite Neoparameoba perurans, the causative agent of AGD, by bath exposure and after 2 weeks, the fish were treated with three commercial PAA products (i.e., Perfectoxid, AquaDes and ADDIAqua) at a dose of 5 ppm. Two exposure durations were evaluated - 30 min and 60 min. Sampling was performed 24 h and 2 weeks after PAA treatment (equivalent to 2- and 4-weeks post infection). At each sampling point, the following parameters were evaluated: gross gill pathology, gill parasitic load, plasma reactive oxygen species (ROS) and total antioxidant capacity (TAC), histopathology and gene expression profiling of genes with key involvement in oxidative stress in the gills and olfactory organ. AGD did not result in systemic oxidative stress as ROS and TAC levels remained unchanged. There were no clear patterns of AGD-mediated regulation of the oxidative stress biomarkers in both the gills and olfactory organ; significant changes in the expression were mostly related to time rather than infection status. However, the expression profiles of the oxidative stress biomarkers in AGD-affected salmon, following treatment with PAA, revealed that gills and olfactory organ responded differently - upregulation was prominent in the gills while downregulation was more frequent in the olfactory organ. The expression of catalase, glutathione S-transferase and thioredoxin reductase 2 was significantly affected by the treatments, both in the gills and olfactory organ, and these alterations were influenced by the duration of exposure and PAA product type. Parasitic load in the gills did significantly increase after treatment regardless of the product and exposure duration; the parasite was undetectable in some fish treated with AquaDes for 30 mins. However, PAA treated groups for 30 min showed lower macroscopic gill scores than the infected-untreated fish. Histology disclosed the classic pathological findings such as multifocal hyperplasia and increased number of mucous cells in AGD-affected fish. Microscopic scoring of gill injuries showed that AGD-infected-PAA-treated fish had lower scores, however, an overall trend could not be established. The morphology and structural integrity of the olfactory organ were not significantly altered by parasitism or PAA treatment. Collectively, the results indicate that AGD did not affect the systemic and mucosal oxidative status of Atlantic salmon. However, such a striking profile was changed when AGD-affected fish were exposed to oxidative chemotherapeutics. Moreover, the gills and olfactory organ demonstrated distinct patterns of gene expression of oxidative stress biomarkers in AGD-infected-PAA-treated fish. Lastly, PAA treatment did not fully resolve the infection, but appeared not to worsen the mucosal health either.

Amebic infections of the central nervous system.

The report of death of a person from amebic meningoencephalitis, the proverbial "brain-eating ameba," Naegleria fowleri, acquired in a state park lake in Iowa in July 2022 has once again raised the seasonal alarms about this pathogen. While exceptionally rare, its nearly universal fatality rate has panicked the public and made for good copy for the news media. This review will address free-living ameba that have been identified as causing CNS invasion in man, namely, Naegleria fowleri, Acanthamoeba species, Balamuthia mandrillaris, and Sappinia diploidea (Table 1). Of note, several Acanthamoeba spp. and Balamuthia mandrillaris may also be associated with localized extra-CNS infections in individuals who are immunocompetent and disseminated disease in immunocompromised hosts. These ameba are unique from other protozoa in that they are free-living, have no known insect vector, do not result in a human carrier state, and are typically unassociated with poor sanitation. Table 1 Free-living ameba that have been identified as causing CNS invasion in man, namely, Naegleria fowleri, Acanthamoeba species, Balamuthia mandrillaris, and Sappinia diploidea Entity Pathogenic ameba Predisposing disorders Portal of entry Incubation period Clinical features Radiographic findings CSF finding Diagnostic measures Primary amebic meningoencephalitis Naegleria fowleri; N. australiensis; N. italica Previously healthy children or young adults Olfactory epithelium 2-14 days (average 5 days) Headache, fever, altered mental status, meningeal signs; seizures Brain edema; meningeal enhancement; hydrocephalus; basal ganglia infarctions Increased opening pressure; neutrophilic pleocytosis (~ 1000 cells/cu mm); low glucose Brain biopsy, CSF wet prep, IIF culture or PCR Granulomatous amebic encephalitis Acanthamoeba spp.; Balamuthia mandrillaris; Sappinia diploidea Typically, immunocompromised individual Skin sinuses; olfactory epithelium respiratory tract Weeks to months Headache; altered mental status seizures, focal neurological findings Focal parenchymal lesions with edema; hemorrhagic infarctions; meningeal enhancement Generally, LP contraindicated; when performed lymphocytic pleocytosis; increased protein; low glucose Brain biopsy, CSF culture, wet prep, IIF, or PCR IIF indirect immunofluorescence, LP lumbar puncture, PCR polymerase chain reaction.