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Naegleria fowleri is the causative agent of primary amoebic meningoencephalitis, a rapid and acute infection of the central nervous system with a fatal outcome in >97% of cases. Due to the infrequent report of cases and diagnostic gaps that hinder the possibility of recovering clinic isolates, studies related to pathogenesis of the disease are scarce. However, the secretion of cytolytic molecules has been proposed as a factor involved in the progression of the infection. Several of these molecules could be included in extracellular vesicles (EVs), making them potential virulence factors and even modulators of the immune response in this infection. In this work, we evaluated the immunomodulatory effect of EVs secreted by two clinic isolates of Naegleria fowleri using in vitro models. For this purpose, characterization analyses between EVs produced by both isolates were first performed, for subsequent gene transcription analyses post incubation of these vesicles with primary cultures from mouse cell microglia and BV-2 cells. Analyses of morphological changes induced in primary culture microglia cells by the vesicles were also included, as well as the determination of the presence of nucleic acids of N. fowleri in the EV fractions. Results revealed increased expression of NOS, proinflammatory cytokines IL-6, TNF-α, and IL-23, and the regulatory cytokine IL-10 in primary cultures of microglia, as well as increased expression of NOS and IL-13 in BV-2 cells. Morphologic changes from homeostatic microglia, with small cellular body and long processes to a more amoeboid morphology were also observed after the incubation of these cells with EVs. Regarding the presence of nucleic acids, specific Naegleria fowleri DNA that could be amplified using both conventional and qPCR was confirmed in the EV fractions. Altogether, these results confirm the immunomodulatory effects of EVs of Naegleria fowleri over microglial cells and suggest a potential role of these vesicles as biomarkers of primary acute meningoencephalitis.

RESUMO

Resumen Las vesículas extracelulares son nanoparticulas secretadas por células procariotas y eucariotas, con funciones variadas que van desde la comunicación intercelular hasta la modulación de la respuesta inmune. La investigación en este tema se enfocó inicialmente en el aislamiento, identificación y caracterización, para luego abarcar los mecanismos fisiológicos en los que se ven involucradas. Más recientemente, la investigación, particularmente centrada en exosomas, ha permitido abrir campo a novedosas hipótesis sobre su utilidad en inmunoterapia y como marcadores biológicos. Esta revisión explora aspectos básicos sobre la biogénesis y la composición de los exosomas, así como su uso en diagnóstico y tratamiento, a partir del conocimiento generado sobre su aislamiento y purificación, distribución de cargos específicos y su relación con la respuesta inmune. Los hallazgos sobre su aplicabilidad en procesos cancerosos son promisorios; sin embargo, existe toda una ventana de posibilidades para investigar esta plataforma molecular como potenciales vacunas acelulares y marcadores de pronóstico, diagnóstico y alerta, tanto en cáncer como en patologías causadas por agentes infecciosos.

Abstract Extracellular vesicles are nanoparticles released by prokaryotic and eukaryotic cells, with a variety of functional roles in intercellular communication and even in modulation of the immune response. Research in this topic was initially focused on isolation, identification and characterization of the vesicles, with subsequent understanding of the physiological mechanisms in which they are involved. Furthermore, recent studies, particularly with exosomes, have opened the field to novel hypotheses about their usefulness in immunotherapy and as biological markers. This review explores general aspects about the biogenesis and composition of exosomes, as well as their potential use in diagnosis and treatment, based on the knowledge generated about their isolation, production, cargoes, delivery engineering and relationship with the immune response. The findings on its applicability in cancerous processes are promising, but there is still a variety of investigation possibilities of this molecular platforms as cell-free vaccines and as prognostic, diagnostic and/or warning markers, both in cancer but also in infectious diseases.

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Extracellular vesicles (EVs) are small lipid vesicles released by both prokaryotic and eukaryotic cells, involved in intercellular communication, immunomodulation and pathogenesis. In this study, we performed a characterization of the EVs produced by trophozoites of a clinical isolate of the free-living amoeba Naegleria fowleri (N. fowleri). Size distribution, zeta potential, protein profile and protease activity were analyzed. Under our incubation conditions, EVs of different sizes were observed, with a predominant population ranging from 206 to 227 nm. SDS-PAGE revealed protein bands of 25 to 260 KDa. The presence of antigenic proteins was confirmed by Western blot, which evidenced strongest recognition by rat polyclonal antibodies raised against N. fowleri in the region close to 80 KDa and included peptidases, as revealed by zymography. Proteins in selected immunorecognized bands were further identified using nano-ESI-MS/MS. A preliminary proteomic profile of the EVs identified at least 184 proteins as part of the vesicles' cargo. Protease activity assays, in combination with the use of inhibitors, revealed the predominance of serine proteases. The present characterization uncovers the complexity of EVs produced by N. fowleri, suggesting their potential relevance in the release of virulence factors involved in pathogenicity. Owing to their cargo's diversity, further research on EVs could reveal new therapeutic targets or biomarkers for developing rapid and accurate diagnostic tools for lethal infections such as the one caused by this amoeba.

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During the first trimester of 2020, the Ministry of Health of Costa Rica reported the first three cases of primary amebic meningoencephalitis (PAM). In two cases, laboratory personnel of the hospitals preliminarily identified amoeboid forms in cerebrospinal fluid (CSF) samples. For the molecular confirmation of species, CSF samples were sent to our laboratory. We carried out microscopic analyses and exflagellation assays. Besides, samples were cultured in 2% casein hydrolysate medium and in non-nutrient agar plates supplemented with Escherichia coli. Finally, PCR and sequencing were employed for the molecular diagnosis and species identification. In all cases, the presence of Naegleria fowleri was confirmed. An environmental investigation to identify the possible infection sources was also performed. Water samples from hot springs and groundwater from an artisan well were collected and after filtration and culture in non-nutrient agar plates supplemented with E. coli, thermotolerance and exflagellation assays were carried out. For the positive samples, PCR and sequencing were performed, confirming the presence of N. fowleri in several water samples. The report of these cases and the possible association with hot springs has had a significant impact on the population and health authorities of Costa Rica.

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Acanthamoeba is a genus of free-living amoebae widely distributed in nature, associated with the development of encephalitis and keratitis. Despite the fact that it is common to find genotype T5 in environmental samples, only a few cases have been associated with clinical cases in humans. The wide distribution of Acanthamoeba, the characteristic of being amphizoic and the severity of the disease motivate researchers to focus on the isolation of these organisms, but also in demonstrating direct and indirect factors that could indicate a possible pathogenic potential. Here, we performed the characterization of the pathogenic potential of an Acanthamoeba T5 isolate collected from a water source in a hospital. Osmo- and thermotolerance, the secretion of proteases and the effect of trophozoites over cell monolayers were analyzed by different methodologies. Additionally, we confirm the secretion of extracellular vesicles (EVs) of this isolate incubated at two different temperatures, and the presence of serine and cysteine proteases in these vesicles. Finally, using atomic force microscopy, we determined some nanomechanical properties of the secreted vesicles and found a higher value of adhesion in the EVs obtained at 37 °C, which could have implications in the parasite´s survival and damaging potential in two different biological environments.

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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.

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BACKGROUND: Acanthamoeba is the genus of free-living amoebae that is most frequently isolated in nature. To date, 20 Acanthamoeba genotypes have been described. Genotype T4 is responsible for approximately 90% of encephalitis and keratitis cases. Due to the ubiquitous presence of amoebae, isolation from environmental sources is not uncommon; to determine the clinical importance of an isolation, it is necessary to have evidence of the pathogenic potential of amoebae. OBJECTIVE: The aim of this study was to physiologically characterise 8 Acanthamoeba T4 isolates obtained from dental units and emergency combination showers and to determine their pathogenic potential by employing different laboratory techniques. METHODS: Eight axenic cultures of Acanthamoeba genotype T4 were used in pathogenic potential assays. Osmotolerance, thermotolerance, determination and characterisation of extracellular proteases and evaluation of cytopathic effects in MDCK cells were performed. FINDINGS: All of the isolates were osmotolerant, thermotolerant and had serine proteases from 44-122 kDa. Two isolates had cytopathic effects on the MDCK cell monolayer. MAIN CONCLUSION: The presence of Acanthamoeba T4 with pathogenic potential in areas such as those tested in this study reaffirms the need for adequate cleaning and maintenance protocols to reduce the possibility of infection with free-living amoebae.

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BACKGROUND Acanthamoeba is the genus of free-living amoebae that is most frequently isolated in nature. To date, 20 Acanthamoeba genotypes have been described. Genotype T4 is responsible for approximately 90% of encephalitis and keratitis cases. Due to the ubiquitous presence of amoebae, isolation from environmental sources is not uncommon; to determine the clinical importance of an isolation, it is necessary to have evidence of the pathogenic potential of amoebae. OBJECTIVE The aim of this study was to physiologically characterise 8 Acanthamoeba T4 isolates obtained from dental units and emergency combination showers and to determine their pathogenic potential by employing different laboratory techniques. METHODS Eight axenic cultures of Acanthamoeba genotype T4 were used in pathogenic potential assays. Osmotolerance, thermotolerance, determination and characterisation of extracellular proteases and evaluation of cytopathic effects in MDCK cells were performed. FINDINGS All of the isolates were osmotolerant, thermotolerant and had serine proteases from 44-122 kDa. Two isolates had cytopathic effects on the MDCK cell monolayer. MAIN CONCLUSION The presence of Acanthamoeba T4 with pathogenic potential in areas such as those tested in this study reaffirms the need for adequate cleaning and maintenance protocols to reduce the possibility of infection with free-living amoebae.

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Angiostrongylus costaricensis is a zoonotic parasitic nematode that causes abdominal or intestinal angiostrongyliasis in humans. It is endemic to the Americas. Although the mitochondrial genome of the Brazil taxon has been published, there is no available mitochondrial genome data on the Costa Rica taxon. We report here the complete mitochondrial genome of the Costa Rica taxon and its genetic differentiation from the Brazil taxon. The whole mitochondrial genome was obtained from next-generation sequencing of genomic DNA. It had a total length of 13,652 bp, comprising 36 genes (12 protein-coding genes-PCGs, 2 rRNA and 22 tRNA genes) and a control region (A + T rich non-coding region). It is longer than that of the Brazil taxon (13,585 bp). The larger mitogenome size of the Costa Rica taxon is due to the size of the control region as the Brazil taxon has a shorter length (265 bp) than the Costa Rica taxon (318 bp). The size of 6 PCGs and the start codon for ATP6, CYTB and NAD5 genes are different between the Costa Rica and Brazil taxa. Additionally, the two taxa differ in the stop codon of 6 PCGs. Molecular phylogeny based on 12 PCGs was concordant with two rRNA, 22 tRNA and 36 mitochondrial genes. The two taxa have a genetic distance of p = 16.2% based on 12 PCGs, p = 15.3% based on 36 mitochondrial genes, p = 13.1% based on 2 rRNA genes and p = 10.7% based on 22 tRNA genes, indicating status of sibling species. The Costa Rica and Brazil taxa of A. costaricensis are proposed to be accorded specific status as members of a species complex.

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

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