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Acanthamoeba genotype T4 keratitis in a patient with Thygeson’s superficial punctate keratitis
(2017-01) Rodríguez Mena, Diego; Retana Moreira, Lissette; Abrahams Sandí, Elizabeth
A case of amoebic keratitis due to Acanthamoeba genotype T4 is reported. The patient is a 74-year old woman with Thygeson’s superficial punctate keratitis. Corneal scrapings were employed for the diagnosis, which included direct microscopic observation, Giemsa stains, cultures in non-nutrient agar and a PCR that amplifies the DF3 fragment of 18S rDNA. The therapeutic use of contact lenses is proposed as the predisposing factor in this case, considering that the patient has a previous damage of the corneal epithelium. This is the first case of amoebic keratitis reported and confirmed in Costa Rica.
Amebas de vida libre como agentes de encefalitis en el ser humano
(2014-07) Abrahams Sandí, Elizabeth; Retana Moreira, Lissette
Este artículo de revisión se enfoca en las amebas de vida libre, un tipo de protozoarios que habitan diferentes tipos de suelo y fuentes de agua. Estos organismos tienen dos estadios evolutivos: trofozoíto y quiste, siendo este último la forma de resistencia que les permite sobrevivir en condiciones adversas del ambiente. La abundancia y diversidad de las amebas de vida libre depende de factores como el clima, pH y disponibilidad de nutrientes. Los trofozoítos se alimentan por fagocitosis y digieren bacterias, hongos y algas ingeridas dentro del fagolisosoma. Además, el artículo señala que algunas de estas amebas tienen la capacidad de comportarse como endoparásitos bajo ciertas condiciones, y que algunas especies de amebas de vida libre pueden ser patógenas para los seres humanos, causando enfermedades como la meningoencefalitis amebiana primaria, la encefalitis granulomatosa amebiana y la queratitis amebiana. Se mencionan algunos casos de infección en humanos y se destaca la importancia de la investigación en este tema para prevenir y tratar estas enfermedades.
Characterization of Extracellular Vesicles Secreted by a Clinical Isolate of Naegleria fowleri and Identification of Immunogenic Components within Their Protein Cargo
(2022-06) Retana Moreira, Lissette; Steller Espinoza, María Fernanda; Chacón Camacho, Natalia; Cornet Gómez, Alberto; Sáenz Arce, Giovanni; Osuna Carrillo de Albornoz, Antonio; Lomonte, Bruno; Abrahams Sandí, Elizabeth
Abstract: Extracellular vesicles (EVs) are small lipid vesicles released by both prokaryotic and eu-karyotic 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 the 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 the 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.
Fatal meningoencephalitis in child and isolation of Naegleria fowleri from hot springs in Costa Rica
(2015-02) Abrahams Sandí, Elizabeth; Retana Moreira, Lissette; Castro Castillo, Alfredo; Reyes Batlle, María; Lorenzo Morales, Jacob
In summary, this investigation identified an N. fowleri ameba in water sources at a resort in Costa Rica that had been visited by a child from the United States who died of PAM as a results of N. fowleri infection. These amebas pose a high risk to human health and were found in an area frequented by tourists, which should alert health authorities in Costa Rica of the need for monitoring locations such as this for possible contamination and notifying the public of the risk for infection.
Futuro de la terapia y el diagnóstico con vesículas extracelulares
(2024) Alvarado Ocampo, Johan; Abrahams Sandí, Elizabeth; Retana Moreira, Lissette
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.
In vitro effects of environmental isolates of Acanthamoeba T4 and T5 over human erythrocytes and platelets
(2020-01-21) Alvarado Ocampo, Johan; Retana Moreira, Lissette; Abrahams Sandí, Elizabeth
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.
Isolation and molecular characterization of Acanthamoeba and Balamuthia mandrillaris from combination shower units in Costa Rica
(2014-08-19) Retana Moreira, Lissette; Abrahams Sandí, Elizabeth; Cabello Vílchez, Alfonso Martín; Reyes Batlle, María; Valladares Hernández, Basilio; Martínez Carretero, Enrique; Piñero Barroso, José Enrique; Lorenzo Morales, Jacob
Free living amoebae (FLA) are ubiquitous protozoa, which may behave as parasites under certain conditions. Four genera are recognized as causal agents of infections in humans and animals: Naegleria, Sappinia, Acanthamoeba and Balamuthia. This work determines the presence of FLA in combination shower units and employs molecular biology for the characterization of isolates. The morphological analy sis and partial sequencing of the 18S rDNA gene revealed the presence of Acanthamoeba genotype T4 in 30 % of the units sampled. In addition to Acanthamoeba cysts, trophozoites with morphological characteristics similar to Balamuthia were identified. PCR assay using the mitochondrial 16S rRNA gene as a target confirmed the identification of the amoeba as Balamuthia mandrillaris. Up to date, this is the first report of the isolation of B. mandrillaris in Central America and the fifth report worldwide.
Isolation and Molecular Characterization of Acanthamoeba Strains from Dental Units in Costa Rica
(2015-03) Retana Moreira, Lissette; Abrahams Sandí, Elizabeth; Castro Artavia, Esteban; Fernández Sánchez, Ana; Castro Castillo, Alfredo; Reyes Batlle, María; Lorenzo Morales, Jacob
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, Balam uthia, and Sappinia. In this study, the presence of Acanthamoeba in dental units was determined and the isolates obtained were molecularly character ized; 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.
Isolation of Acanthamoeba T5 from Water: Characterization of Its Pathogenic Potential, Including the Production of Extracellular Vesicles
(2020-01-23) Retana Moreira, Lissette; Vargas Ramírez, Daniel; Linares Ordoñez, Fátima; Prescilla Ledezma, Alexa; Vaglio Garro, Annette; Osuna Carrillo de Albornoz, Antonio; Lorenzo Morales, Jacob; Abrahams Sandí, Elizabeth
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.
Overview of extracellular vesicles in pathogens with special focus on human extracellular protozoan parasites
(2024) Alvarado Ocampo, Johan; Abrahams Sandí, Elizabeth; Retana Moreira, Lissette
Extracellular vesicles (EVs) are lipid-bilayered membrane-delimited particles secreted by almost any cell type, involved in different functions according to the cell of origin and its state. From these, cell to cell communication, pathogen-host interactions and modulation of the immune response have been widely studied. Moreover, these vesicles could be employed for diagnostic and therapeutic purposes, including infections produced by pathogens of diverse types; regarding parasites, the secretion, characterisation, and roles of EVs have been studied in particular cases. Moreover, the heterogeneity of EVs presents challenges at every stage of studies, which motivates research in this area. In this review, we summarise some aspects related to the secretion and roles of EVs from several groups of pathogens, with special focus on the most recent research regarding EVs secreted by extracellular protozoan parasites.
Potentially pathogenic Acanthamoeba genotype T4 isolated from dental units and emergency combination showers
(2017-12) Castro Artavia, Esteban; Retana Moreira, Lissette; Lorenzo Morales, Jacob; Abrahams Sandí, Elizabeth
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
Primary Amebic Meningoencephalitis Related to Groundwater in Costa Rica: Diagnostic Confirmation of Three Cases and Environmental Investigation
(2020-08-01) Retana Moreira, Lissette; Zamora Rojas, Leidy; Grijalba Murillo, Muriel; Molina Castro, Silvia Elena; Abrahams Sandí, Elizabeth
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.
Primer aislamiento de Balamuthia mandrillaris en Costa Rica
(2014-07) Retana Moreira, Lissette; Abrahams Sandí, Elizabeth
En este estudio se determinó la presencia de Balamuthia mandrillaris en una muestra de polvo procedente de una unidad combinada de emergencia en Costa Rica. Se realizó un muestreo de polvo y agua en 12 unidades combinadas de emergencia instaladas en laboratorios de docencia. Las muestras fueron cultivadas en agar no nutritivo al 1,5% suplementado con Escherichia coli. Las amebas de vida libre aisladas a partir de estas muestras se identificaron morfológicamente y se realizó la caracterización molecular empleando una PCR que amplifica el gen mitocondrial 16S ARNr. La secuenciación del producto de PCR obtenido confirmó el aislamiento de B. mandrillaris, una ameba altamente patógena que produce principalmente cuadros a nivel de sistema nervioso central y piel. A pesar de que el aislamiento de esta especie no se ha ligado a ningún caso clínico en Costa Rica, este reporte es de gran importancia pues representa el primer aislamiento de B. mandrillaris en América Central y el quinto aislamiento a nivel mundial a partir de una muestra ambiental.
Primer aislamiento e identificación molecular de Naegleria fowleri en Costa Rica
(2015-01) Abrahams Sandí, Elizabeth; Retana Moreira, Lissette
La meningoencefalitis amebiana primaria, causada por Naegleria fowleri, es un cuadro agudo y fulminante. Esta es una ameba de vida libre (AVL) que presenta amplia distribución a nivel mundial. Presenta una moratildad alta (cerca del 95%) en la mayoría de los casos diagnosticados alrededor del plantea. La Sección de Protozoología Médica de la Faculta de Microbiología fue contactada por el Ministerio de Salud debida a la presencia de una AVL en las aguas termales de la zona de San Carlos, Alajuela
Providing an in vitro depiction of microglial cells challenged with immunostimulatory extracellular vesicles of Naegleria fowleri
(2024) Retana Moreira, Lissette; Cornet Gómez, Alberto; Sepúlveda Justo, María del Rosario; Molina Castro, Silvia Elena; Alvarado Ocampo, Johan; Chaves Monge, Frida; Jara Rojas, Mariana; Osuna Carrillo de Albornoz, Antonio; Abrahams Sandí, Elizabeth
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.

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Examinando Salud por Autor "Abrahams Sandí, Elizabeth"

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