Identificación de amebas de vida libre en tanques de almacenamiento de agua de edificios del área metropolitana de Ambato / Identification of free-living amoebas in water storage tanks of buildings in the metropolitan area of Ambato

Naegleria fowleri, Acanthamoeba spp. y Balamuthia mandrillaris, son especies de ameba de vida libre (AVL) que causan enfermedades en el sistema nervioso central grave y puede ser fatal en humanos y otros animales. Todos los géneros de AVL tienen dos etapas: quiste y trofozoíto. Los trofozoítos se dividen por fisión binaria, se alimentan activamente y es la etapa infecciosa del organismo. En el medio ambiente, los acanthopodios permiten que los trofozoítos se adhieran a las superficies de las bacterias, hongos, algas y detritos en los que se encuentran su alimento. Acanthamoeba spp. Parecen mantener una relación endosimbiótica con algunas de las bacterias que consumen. Muchas de estas bacterias, como la Legionella, Pseudomonas, Vibrio y algunas micobacterias parecen adquirir factores de virulencia mientras están viviendo dentro de las amebas, lo que les permite sobrevivir y prosperar dentro de otras células fagocíticas (por ejemplo, macrófagos humanos). La tenacidad de AVL para proliferar en ecosistemas hostiles y sobrevivir a la desecación o desinfección hace de estos organismos una fuente potencial transmitida por el agua, por los alimentos y el aire. Conocidos por su ubicuidad, los AVL han sido aislados de diversas matrices ambientales, como suelo, agua dulce y salobre, aguas residuales, aguas termales, polvo y otros. En función de lo antes comentado, este trabajo tiene como objetivo identificar amebas de vida libre en tanques de almacenamiento de agua de edificios del área metropolitana de la ciudad de Ambato (Ecuador), con el fin de aplicar soluciones correctivas en cualquiera de sus puntos. Según el sitio de muestreo, el género de AVL identificado con mayor frecuencia en la superficie fue Balamuthia sp (70,59%) y Acanthamoeba sp., con un 37,78 %. El fondo estuvo caracterizado por Naegleria sp. con un 44,44%(AU)

Naegleria fowleri, Acanthamoeba spp. and Balamuthia mandrillaris are species of free-living amoeba (FLA) that cause severe central nervous system disease and can be fatal in humans and other animals. All genera of FLA have two stages: cyst and trophozoite. Trophozoites divide by binary fission, actively feed, and is the infective stage of the organism. In the environment, acanthopods allow trophozoites to adhere to the surfaces of bacteria, fungi, algae, and detritus on which their food is found. Acanthamoeba spp. they appear to maintain an endosymbiotic relationship with some of the bacteria they consume. Many of these bacteria, such as Legionella, Pseudomonas, Vibrio, and some mycobacteria appear to acquire virulence factors while living inside amoebas, allowing them to survive and thrive inside other phagocytic cells (eg, human macrophages). FLA's tenacity to proliferate in hostile ecosystems and survive desiccation or disinfection makes these organisms a potential waterborne, foodborne, and airborne source. Known for their ubiquity, FLAs have been isolated from various environmental matrices, such as soil, fresh and brackish water, wastewater, hot springs, dust, and others. Based on the aforementioned, this work aims to identify free-living amoebas in water storage tanks of buildings in the metropolitan area of the city of Ambato (Ecuador), in order to apply corrective solutions at any of its points. According to the sampling site, the FLA genus most frequently identified on the surface was Balamuthia sp (70.59%) and Acanthamoeba sp., with 37.78 %. The background was characterized by Naegleria sp. with 44.44%(AU)

Isolamento de amebas de vida livre potencialmente patogênicas em piscinas de uso coletivo localizadas no município de Redenção, Pará, Brasil / Isolation of potentially pathogenic free-living amoebae in swimming pools for collective use located in the municipality of Redenção, Pará, Brazil

Objetivo Isolar amebas dos gêneros Acanthamoeba e Naegleria em piscinas de uso coletivo do município de Redenção, Pará, Brasil. Métodos A identificação dessas amebas se deu a partir de amostras retiradas de piscinas de uso coletivo de quatro clubes da cidade. As análises foram realizadas a partir do exame direto a fresco, cultura, análise morfológica após coloração de Gram e teste de exflagelação, seguindo o protocolo descrito por Carlesso, Artuso e Rott. Resultados As amebas de vida livre foram encontradas em 41,67% das amostras, com predomínio das do gênero Acanthamoeba (63,2%) no exame direto a fresco. No clube A, localizado no setor Serrinha, houve predominância dessas amebas em relação às demais (8,34%). As amostras obtidas por swabs apresentaram 73,69% de formas evolutivas em comparação à análise feita apenas na água das piscinas. A confirmação dos gêneros das amebas presentes nas amostras feita através da coloração de Gram encontrou um perfil de identificação diferente, sendo que nesse exame a ameba de gênero Naegleriaspp. se sobressaiu perante a de gênero Acanthamoeba (61,11% e 38,89%, respectivamente). Conclusão Os resultados do estudo confirmam a presença de isolados potencialmente patogênicos de amebas de vida livre em piscinas de uso coletivo, o que pode significar um risco à saúde pública.

Objective To isolate amoebae of the genera Acanthamoeba and Naegleria in swimming pools for collective use in the municipality of Redenção, Pará, Brazil. Methods The identification of these amoebae was determined from samples of swimming pools for collective use in four private clubs in the city. The analysis methodology was performed based on direct fresh examination, culture, morphological analysis after Gram staining and exflagellation test, following the protocol described by Carlesso, Artuso and Rott. Results The frequency of free-living amoebae was 41.67%, with a predominance of the Acanthamoeba genus (63.2%) in direct fresh examination. At club A, located in the Serrinha sector, the frequency of these amoebae was higher than the others (8.34%). The samples obtained by swabs showed 73.69% of evolutionary forms in comparison to the analysis only of the water in the pools. The confirmation of the genera by Gram stain showed a different identification profile, in which Naegleria spp. stood out before the genus Acanthamoeba (61.11% and 38.89%, respectively). Conclusion In conclusion, the results of the study confirm the presence of potentially pathogenic isolates from free-living amoebae in swimming pools for collective use that may present risks to public health.

Acanthamoeba spp. y Naegleria spp. aisladas del Río Pamplonita, zona metropolitana de Cúcuta, Colombia / Acanthamoeba spp. and Naegleria spp. isolated from Pamplonita river, metropolitan area of Cucuta, Colombia

Introducción: Acanthamoeba y Naegleria son géneros de amebas de vida libre resistentes a cambios extremos de temperatura y pH, aislados de diversos ambientes (suelo, aire y agua). Debido a la cantidad de habitantes que se benefician de las aguas del Río Pamplonita, al desconocimiento de su presencia en la región y al aumento de enfermedades relacionadas con el consumo de agua, se considera necesario realizar estudios sobre la distribución de estos microorganismos. Objetivo: Identificar Acanthamoeba spp. y Naegleria spp. en aguas del curso principal del río Pamplonita de la zona metropolitana de Cúcuta, Colombia. Métodos: La búsqueda de las amebas de interés se realizó mediante examen directo y cultivo a 28, 37 y 42 °C en agar no nutritivo a partir de 21 muestras de agua de siete sectores del río Pamplonita. Adicionalmente, se midió pH y temperatura in situ y se determinó la carga de coliformes. Resultados: Se encontró que 76,2 por ciento de las muestras fueron positivas para alguna de las amebas. La Acanthamoeba fue la que se aisló con mayor frecuencia. El 28,6 por ciento de los cultivos incubados a 42 °C fueron positivos principalmente para Naegleria spp., lo que indica que estos aislados termotolerantes podrían tener potencial patógeno. Conclusiones: Acanthamoeba spp. y Naegleria spp. son frecuentes en aguas del Río Pamplonita en áreas de importante intervención humana. El hallazgo de amebas termotolerantes alerta sobre el riesgo de salud para la población que se encuentra expuesta a esta fuente hídrica(AU)

Introduction: Acanthamoeba and Naegleria are genus of free-living amoeba (AVL) resistant to extreme changes in temperature and pH, isolated from different environments (soil, air and water). Due to the number of inhabitants that benefit from the waters of Pamplonita River, the lack of knowledge about its presence in the region and the increase of diseases related to water consumption, it is necessary to carry out studies on its distribution. Objective: To identify Acanthamoeba spp and Naegleria spp in the water of the main course of Pamplonita River, in the metropolitan area of Cúcuta, Colombia. Methods: The search for the amoebas of interest was performed by direct examination and culture at 28, 37 and 42 °C on non-nutritive agar from 21 water samples from seven sectors of Pamplonita River. Additionally, pH and temperature were measured in situ and the coliforms´ load was also determined. Results: It was found that 76.2 percent of the samples were positive for some of the amoebas, with Acanthamoeba being the most frequently isolated. 28.6 percent of the cultures incubated at 42 °C were positive mainly for Naegleria spp, indicating that these thermotolerant isolates could have pathogen potential. Conclusions: Acanthamoeba spp and Naegleria spp are frequent in waters of Pamplonita River which are areas of important human intervention. The finding of thermotolerant amoebas warns about the health risk for the population that is exposed to this water source(AU)

Occurrence of thermotolerant hartmannella vermiformis and naegleria spp. in hot springs of Ardebil province, northwest Iran

Geothermal waters could be suitable niches for thermophilic free living amoebae including Naegleria and Hartmannella. Ardebil Province, northwest Iran is popular for having many hot springs for recreational and health purposes activity. The present research is the first molecular based investigation regarding the presence of Naegleria and Hartmannella in the hot springs of Ardebil Province in Iran. Overall, 30 water samples were taken from waters of thermal hot springs in Ardebil Province, Iran during 2010-2011. All collected samples were transferred to Dept. of Parasitology and Mycology, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Cultivation of concentrated water samples was performed using culture-enrichment method. Cloning of the target amoebae was obtained and morphological and molecular analysis was done using page key combined with two sets of primers, respectively. Sequence analysis and homology search was used for strains identification. Of 30 water samples, 8 [26.7%] were positive for thermotolerant Vahlkampfiids and Hartmannella based on morphological characteristics of vegetative form and double walled cysts. Cloning of the target amoebae were done successfully. Sequencing of the positive isolates revealed that the strains belonged to Naegleria [N. carteri and N. spp] and H. vermiformis. The result highlights a need for improved filtration and disinfection and periodic monitoring of recreational thermal waters in order to prevent disease related to free- living amoebae. This is the first comprehensive molecular study of thermophilic Naegleria and Hartmannella in hot springs of Iran

Cepas de Naegleria aisladas en santiago de Cuba y estudiadas a través del microscopio electrónico de barrido / Naegleria strains isolated in Santiago de Cuba and studied using the scanning electron microscope

A partir de 4 localidades donde se aislaron amebas del género Naegleria fowleri en Santiago de Cuba, se realizaron las técnicas necesarias para su adecuado estudio a través del microscopio electrónico de barrido. Con anterioridad, esas amebas fueron inoculadas en biomodelos experimentales (ratones), lo cual permitió determinar un nivel de patogenicidad diferente; y esto último, como es un aspecto controvertido en diversas teorías sobre sus causas (algunas analizadas aquí), entre las que se incluye la sustentada en la invaginación de su superficie (amebostomas o suctores), la autora de este trabajo se dio a la tarea de valorar la presencia (número y características) de los amebostomas o su ausencia a través del mencionado aparato, de donde se derivó que los ameboflagelados no patógenos apenas se diferenciaban de los que sí lo eran. Tal hallazgo pudo ser reafirmado cuando estos se compararon con 2 cepas reconocidas en el Instituto de Medicina Tropical de Checoslovaquia; por consiguiente, ese resultado descarta la citada causa en la patogenicidad de las amebas.

Taking into account 4 localities where amebas of the genus Naegleria fowleri were isolated in Santiago de Cuba, necessary techniques for their adequate study using the scanning electron microscope were performed. Previously, those amebas were inoculated in experimental biomodels (mice), which allowed to determine a different level of pathogenicity, a controversial issue in several theories about their causes (some discussed here), including that maintained in their surface invagination (amebostomas or suckers). Thus, the author of this work evaluated the presence (number and characteristics) or absence of amebostomas through such device, and it is concluded that pathogenic ameboflagellates were similar to those that were not. This finding could be confirmed when they were compared with 2 strains recognized in the Institute of Tropical Medicine of Czechoslovakia. Consequently, that result rules out the above cause in the pathogenesis of amebas.

Amibas de vida libre Acanthamoeba, Balamuthia, Naegleria, Sappinia y Paravahlkampfia: agentes de infección en humano y transmisoras de microorganismos / Free-living amoebae Acanthamoeba, Balamuthia, Naegleria, and Paravahlkampfia Sappinia: agents of human infection and transmission of microorganisms

Se han reportado como agentes causales de enfermedad en humanos: Naegleria fowleri, agente causal de meningoencefalitis amibiana primaria en individuos inmunocompetentes: varias especies de género Acanthamoeba que pueden producir encefalitis granulomatosa amibiana en individuos inmunosuprimidos, úlceras corneales, lesiones en piel y otros órganos en personas inmunocompetentes; Ballamuthia mandrillares agente causal de encefalitis y lesiones en piel en personas inmunocompetentes, Sappinia pedata responsable de un caso de encefalitis en un individuo inmunocompetente y Paravahlkamphfia sp en un caso de queratitis y Paravahlkamphfia francinae en un caso de meningoencefalitis aminiana primera, en paciente en paciente inmunocompetente. No se conoce totalmente los factores de los que depende que las enfermedades producidas por amibas de vida libre se presenten, influyen la presencia de algunas enfermedades, contacto con aguas en las cuales se encuentran estas amibas, y la presencia de enzimas capaces de ejercer efecto citopático. Además el uso de lentes de contacto, algunas costumbres y necesidades humanos rompen el equilibrio ecológico, aumentando así la probabilidad de contacto del hombre con estas amibas. Se han evidenciado en ellas microorganismo en un proceso de endosimbiosis lo que por lo que amibas de vida libre intervienen en la transmisión de ciertos microorganismos patógenos y las amibas se transforman en un incubador potencial y transmisorasde agentes infecciosos. Debe investigarse sobre aspectos biológicos y la relación con el humano.

Have been reported as causative agents of disease in humans: Naegleria fowleri, the causative agent of primary amoebic meningoencephalitis in immunocompetent individuals, several species of Acanthamoeba that con cause granulomatous amoebic encephalitis in immunosuppressed individuals, corneal ulcers, skin lesions and other organs in people immunocompetent; Ballamuthia madrillares causative agent of encephalitis and skin lesions in immunocompetent persons, Sappinia pedata, responsible for a case of encephalitis in an immunocompetent individual Paravahlkamphfia sp in a case of keratitis and Paravanhlkamphfia francinae in a case of primary amoebic meningoencephalitis in an immunocompetent patient. Not fully understood the factors that it depends on diseases caused by free-living amoebae are present, influencing the presence of some diseases, contact with water where are these amoebae, and the presence of enzymes capable of the cytopathic effect. Furthermore, the use of contact lenses, some customs and human needs break the ecological balance, thereby increasing the likelihood of human contact with these amoebae. Have been evidenced in these microorganisms in the process of endosymbiosis that so the free-living amoebae in the transmission of certain pathogenic microorganisms and amoebas and become a potential incubator and transmission of infectious agents. Biological aspects should be investigated and the relationship with the human.

The nfa1 Gene Contributed on the Contact-dependent Pathogenic Mechanisms of Naegleria fowleri / 한양의대학술지

Free-living Naegleria fowleri is a causal agent of primary amoebic meningoencephalitis in mainly children and young adults. An nfa1 gene, encoding 360 bp of nucleotides, was cloned from a N. fowleri cDNA library by SEREX method. By immunohistochemistry and a confocal microscope, Nfa1 protein was found in amoebic pseudopods, especially in food-cups, when amoeba was in contact with target cells. When an anti-Nfa1 antibody was added to the coculture system, the cytotoxicity of N. fowleri trophozoites onto target cells was decreased, and the severe morphological destruction of rat microglial cells cocultured with N. fowleri trophozoites was reduced. In a tansfection system, an expression vector with an nfa1 gene was successful transfected into nonpathogenic N. gruberi, and transgenic N. gruberi showed the increasing in vitro cytotoxicity. The siRNA decreased the expression levels of nfa1 mRNA and Nfa1 protein in transfected N. fowleri trophozoites. On the immunization of mice with the rNfa1 protein, the protective immunity of host was induced. Thus, mice showed the prolonged mean survival times in PAM-developed mice. In final, the nfa1 gene and Nfa1 protein play an important role in the pathogenesis of N. fowleri infection.

Prevalence of potentially pathogenic free-living amoebae from Acanthamoeba and Naegleria genera in non-hospital, public, internal environments from the city of Santos, Brazil

Acanthamoeba and Naegleria species are free-living amoebae (FLA) found in a large variety of natural habitats. The prevalence of such amoebae was determined from dust samples taken from public non-hospital internal environments with good standards of cleanliness from two campuses of the same University in the city of Santos (SP), Brazil, and where young and apparently healthy people circulate. The frequency of free-living amoebae in both campuseswas 39 percent and 17 percent respectively, with predominance of the genus Acanthamoeba. On the campus with a much larger number of circulating individuals, the observed frequency of free-living amoebae was 2.29 times larger (P< 0.00005). Two trophozoite forms of Naegleria fowleri, are the only species of this genus known to cause primary amoebian meningoencephalitis, a rare and non-opportunistic infection. We assume that the high frequency of these organisms in different internal locations represents some kind of public health risk.

Isolation of vahlkampfids [willaertia magna] and thecamoeba from soil samples in Iran

Vahlkampfiids contains wide variety of genuses with some known as human pathogens such as Naegleria, Vahlkampfia and non pathogens such as Willaertia. Since there was no evidence of presence of Vahlkampfiids in different sources in Iran, we have analyzed soil samples to clarify the presence of these amebas. Seven soil samples collected in Tehran were analyzed to clarify the presence of Vahlkampfiids in soil sources, using microscopic examination of non nutrient agar cultures and specific Vahlkampfiids primer pair. Vahlkampfiids were detected in 2 out of 7 soil samples by direct examination of cultures. Sequence analysis confirmed that Willaertia magna [W. magna] was present in 2 samples. Additionally, Thecamoeba were detected in all of soil samples. To the best of our knowledge, this is the first report of existing W. magna and Thecamoeba in Iran. Over all, more research should be implicated in Iran for identification of Vahlkampfiids within different environmental sources as well as their pathogenic capability relevant for human beings

Free-living Acanthamoeba and Naegleria spp. amebae in water sources of León, Nicaragua

free-living amebae (FLA) are known to occur worldwide in water-related biotopes, but only limited information is available on these organisms in developing countries and so far no information on their presence is available from Nicaragua. The aims of this study were to evaluate the prevalence of potentially pathogenic Acanthamoeba spp. and Naegleria spp. in different water sources to which the population of León municipality is exposed. Since pathogenic amebae are thermotolerant, we were especially interested in the occurrence of FLA in geothermal areas. Water samples were collected from León area in Nicaragua: 88 samples were from rivers and springs, 111 from wells, 74 from water taps and 21 from water tanks in urban and suburban León and from three nearby geothermal areas of San Jacinto, Posoltega and Tipitapa. Amebae were identified using morphological and physiological criteria, immunohistochemical staining procedures and molecular methods. Indirect immunofluorescent test was performed on cysts and trophozoites fixed on microscopical slides and incubated for 30 min at room temperature in separate experiments with the following antibodies: rabbit-anti N. fowleri/N. lovanensis (Nf-Pab), mouse monoclonal antibody anti N. fowleri (Nf-5D12u), rabbit antibodies against Acanthamoeba spp. And fluorescent in situ hybridization (FISH) was performed using 18S rRNA-targeted fluorescent oligonucleotide probes. Probes: GSP for the detection of Acanthamoeba and NAEG1088 for the detection of Naegleria. Free-living amebae were recovered from approximately 43 % of the samples. Acanthamoeba spp was found in 21 % of samples from León municipality and in 2 % of samples from geothermal areas. Amoeboflagellates were found in 10 % of samples from León and in 19 % in geothermal areas. Fifty three percent of tested wells in the geothermal area contained thermotolerant amoeboflagellates. Naegleria spp. was identified in 24 out of 39 (61.5 %) of isolated amoeboflagellates. Twelve of them were assigned to N. lovanenesis while none of the isolates could be identified as N. fowleri. However, the common presence of thermotolerant Naegleria in water, specially N. lovanensis, which is an indicator species for N. fowleri, suggests that also this pathogenic amoeba may pose a risk to public health in the area. On the other side, direct pathogenicity, free-living amebae are receiving increasing attention as reservoirs and potential vehicles for the transmission of bacteria in the environment. Thus the information provided in this study may serve as base-line for future studies on the role of free-living amebae e.g. in waterborne-disease outbreaks in the region. Among such potentially important enteropathgens are Vibrio cholerae, E. coli 0157, and Helicobacter pylori. Rev. Biol. Trop. 56 (2): 439-446. Epub 2008 June 30.

Las amebas de vida libre (AVL) son un grupo de organismos de distribución mundial. Entre las AVL hay parásitos facultativos en humanos y otros animales, los cuales pertenecen a los géneros Acanthamoeba, Naegleria y Balamuthia que causan infecciones severas en el sistema nervioso central. Sin embargo no se tiene ninguna información de Nicaragua. El objetivo de este estudio fue evaluar la presencia de amebas de vida libre, en diferentes fuentes de agua de la parte urbana y rural del Departamento de León, y áreas geotérmicas de Nicaragua. Estas amebas fueron identificadas usando criterios morfológicos, fisiológicos, histoquímicos y moleculares. En los resultados se encontró amebas de vida libre en el 43% del total de las muestras. En la municipalidad de León, se encontraron 21% de Acanthamopeba sp. y en las áreas geotérmicas un 2%. Las amebaflagelados tipo Naegleria fueron 10% y 19% respectivamente. Del grupo amebaflagelados fueron 24, de las cuales 12 se dentificaron como N. lovanensis. En estas muestras no se aisló N. fowleri (ameba patógena), sin embargo, la presencia de N. lovanensis es in indicador de la presencia de N. fowleri, la cual puede ser un riesgo a la salud pública. Además, estas amebas pueden servir como vectores de bacterias enteropatógenas.

Patogenicidad de las amebas de vida libre aisladas de fuentes de agua en Lima / Patogenicity of free-living amoebas isolates in body waters from Lima

Objetivos. Determinar la presencia de amebas de vida libre (AVL) en fuentes de agua del departamento de Lima y evaluar su capacidad patógena en ratones normales e inmunosuprimidos. Materiales y métodos. Se recolectaron muestras de agua de ríos, lagos, piscinas y pozos de Lima. El aislamiento se realizó por cultivo en agar no nutritivo al 2 por ciento con E. coli o E. aerogenes a 37 °C. Posteriormente se instiló en ratones normales e inmunosuprimidos (betametasona 1,4 mg/mL, dosis única) una suspensión con las amebas aisladas. A los 15 díasse evaluaron histológicamente los daños en ambos grupos. Resultados. Se identificaron AVL en 40/83 muestras, 31,3 por ciento de las muestras se desarrollaron en los cultivos. Se aislaron 26 cepas de AVL de siete géneros (Hartmannella, Acanthamoeba, Mayorella, Naegleria, Vahlkampfia, Vannella y Saccamoeba), Acanthamoeba fue la más frecuente (44,2 por ciento). Se encontraron AVL en 53,8 por ciento de los ratones inmunosuprimidos y 15,4 por ciento de los normales (p menor que 0,05). Conclusiones. Existen AVL en cuerpos de agua de Lima, las cuales tienen un potencial patógeno en pacientes inmunosuprimidos, existiendo un riesgo de infección amebiana asociada con estas fuentes de agua.

Objectives. To determine the presence of free-living amoeba (FLA) in water bodies from Lima department and assess their pathogenic ability in normal mice and immunosuppressed. Material and methods. Water samples were collected from rivers, lakes, swimmingpools and wells in Lima, Peru. The isolation was performed by culture in non-nutritious agar with 2 per cent of E. coli or E. aerogenes at 37 °C. Subsequently were injected into normal mice and immunosuppressed (betamethasone 1.4 mg/mL, a single dose) with a suspension of amoeba isolated. For the 15 days were assessed histological damage in both groups. Results. AVL were identified in 40/83 samples, 31.3 per cent of the samples were developed in cultures. 26 strains were isolated from AVL of seven genera (Hartmannella, Acanthamoeba,Mayorella, Naegleria, Vahlkampfia, Vannella and Saccamoeba), Acanthamoeba was the most frequent (44.2 per cent). AVL was found in 53.8 per cent of immunosuppressed mice and 15.4 per cent of the normal (p minor that 0.05). Conclusions. AVL exist in water bodies from Lima, which have a potential pathogen in immunosuppressed patients, there is a risk of amoebic infection associated with these water sources.

Amebas de vida libre en lesiones cutáneas: reporte de 4 casos / Free-living amoebae in skin lesions: report of 4 cases

Las amebas de vida libre son organismos anfizoicos que afectan al hombre produciendo diversas patologías. Los géneros de importancia son Acanthamoeba, Naegleria, Balamuthia y recientemente Sappinia. Se describen cuatro casos de compromiso cutáneo por amebas de vida libre del género Acanthamoeba tres pacientes inmunocomprometidos y uno inmunocompetente. Los inmunocompremetidos presentan lesiones ulcerativas necrotizantes que no llegan a alcanzar al sistema nervioso central. El paciente inmunocompetente presentó un tumor en párpado inferior derecho e hizo compromiso neurológico severo de evolución fatal.

Spa, springs and safety.

Natural mineral water has long been used worldwide for bathing and health purposes. At present, Thailand is famous for health spas and natural hot springs among local people and tourists. Due to possible risks of exposure to harmful agents, we studied hazardous pollutants at 57 natural hot springs from 11 provinces in northern, central, eastern and southern Thailand. Pathogenic, free-living amebae of the genera Naegleria and Acanthamoeba, which can cause central nervous system infection, were found in 26.3% (15/57) and 15.8% (9/ 57), respectively. Dissolved radon, a soil gas with carcinogenic properties, was present in nearly all hot springs sites, with concentration ranging from 0.87-76,527 Becquerels/m3. There were 5 water samples in which radon concentration exceeded the safety limit for drinking. Legionella pneumoniphila (serogroups 1, 3, 5, 6, 7 10 and 13) were found in samples from 71.9% (41/57) of studied sites. Because spas and natural springs are popular tourist attractions, health authorities should be aware of possible hazards and provide tactful measures and guidelines to ensure safety without causing undue alarm to foreign and Thai tourists.

Free-living ameba contamination in natural hot springs in Thailand.

Thermo tolerant free-living ameba, Naegleria spp and Acanthamoeba spp contamination in natural hot springs in Thailand were carried out from 13 provinces. The temperature of hot springs water varied from 28 degrees-65 degrees C and pH from 6-8. We found that 38.2 % (26/68) of water samples were positive, Acanthamoeba was 13.2% (9/68) whilst Naegleria was 35.3% (24/68). Contamination by free-living ameba in natural hot springs may pose a significant health risk to people who use such water for recreation activities.

Early detection and identification of amphizoic amoebae from nasal exudates of a symptomatic case.

A man visited the Out Patient Department of the hospital for Tropical Diseases in February 2004 with low grade fever and severe headache for a week. He had the history of diving in a natural pond 2-3 days before the onset of the disease. A thick bloody mucous was observed from the nasal discharge. Fresh microscopic observation of the exudates in 0.85% sodium chloride revealed numerous active amoeba trophozoites. Two groups of the trophozoites were observed The first group was 10 micro sized amoeba with active directional movement by lobopodia and the second group was 15-30 micro sized amoeba with active multiprogressive movement by filopodia. Few flagellate forms were observed after exflagellation in distilled water and some polygonal cysts were also found. Giemsa' stain was used to differentiate the amoeba trophozoites from the leukocytes. It was concluded that this patient was infected by both Naegleria spp. and Acanthamoeba spp. This is the first report of double infection of free-living amoeba in a symptomatic and non-fatal patient.

Pathogenic free-living amoebae in Korea

Acanthamoeba and Naegleria are widely distributed in fresh water, soil and dust throughout the world, and cause meningoencephalitis or keratoconjunctivitis in humans and other mammals. Korean isolates, namely, Naegleria sp. YM-1 and Acanthamoeba sp. YM-2, YM-3, YM-4, YM-5, YM-6 and YM-7, were collected from sewage, water puddles, a storage reservoir, the gills of a fresh water fish, and by corneal washing. These isolates were categorized into three groups based on the mortalities of infected mice namely, highly virulent (YM-4), moderately virulent (YM-2, YM-5 and YM-7) and nonpathogenic (YM-3). In addition, a new species of Acanthamoeba was isolated from a freshwater fish in Korea and tentatively named Korean isolate YM-4. The morphologic characters of its cysts were similar to those of A. culbertsoni and A. royreba, which were previously designated as Acanthamoeba group III. Based on experimentally infected mouse mortality, Acanthamoeba YM-4 was highly virulent. The isoenzymes profile of Acanthamoeba YM-4 was similar to that of A. royreba. Moreover, an anti-Acanthamoeba YM-4 monoclonal antibody reacted only with Acanthamoeba YM-4, and not with A. culbertsoni. Random amplified polymorphic DNA marker analysis and RFLP analysis of mitochondrial DNA and of a 18S small subunit ribosomal RNA, placed Acanthamoeba YM-4 in a separate cluster based on phylogenic distances. Thus Acanthamoeba YM-4 was identified as a new species, and assigned Acanthamoeba sohi. Up to the year 2002 in Korea, two clinical cases were found to be infected with Acanthamoeba spp. These patients died of meningoencephalitis. In addition, one case of Acanthamoeba pneumonia with an immunodeficient status was reported and Acanthamoeba was detected in several cases of chronic relapsing corneal ulcer, chronic conjunctivitis, and keratitis.

Studies on the biology and pathogenicity of free living amoebae in Sohag

Naegleria and Acanthamoeba are facultative parasites that cause mild to severe meningoencephalitis. In this study, samples were randomly collected from different water and soil sources in Sohag City and cultured on non-nutrient agar medium previously seeded with Escherichia coli. The morphological characters of the isolated amoebae were studied using different stains. The pathogenicity of the isolated amoebae was studied through inoculation into albino rats and rabbits. Rat inoculation with Naegleria trophozoites resulted in a severe histopathological effect on brain tissue, while Acanthamoeba failed to induce infection. Acanthamoeba keratitis could be established in the cornea of rabbits

Effect of antifungal drugs on pathogenic Naegleria spp isolated from natural water sources.

Five of 16 strains of pathogenic Naegleria spp isolated from 350 natural water sources in Taling Chan District, Bangkok had similar molecular weights and zymogram patterns to those of Naegleria fowleri CDCVO 3081 and Thai strain. The in vitro effects of antifungal drugs (amphotericin B, ketoconazole, fluconazole and itraconazole) were tested at the following concentrations: amphotericin B 0.01-0.55 microg/ml, ketoconazole 0.01-0.3 microg/ml, fluconazole 0.75-3.5 mg/ml and itraconazole 4-12 mg/ml respectively. Aliquots (15,000 cells/ml) of the amoebae were placed in the cells of the microtiter plate and incubated at 37 degrees C. Amoebae from each treatment sample were exposed to one of the four antifungal drugs. Statistical analysis was done by dependent t-test. The sensitivity of the antifungal drugs (MIC50) was as follows: amphotericin B 0.03-0.035 microg/ml ketoconazole 0.05-0.15 microg/ml fluconazole 1.75 mg/ml and itraconazole 8-9 mg/ml respectively (p < 0.005). CONCLUSION: Amphotericin B and ketoconazole are more active against Naegleria fowleri in vitro. The results of the present study should be used as an in vitro screening test for drugs that have potential amebicidal activity.

Isolamento de amebas de vida livre potencialmente patogênicas em poeira de hospitais / Isolation of potencially pathogenic free-living amoebas in hospital dust

OBJETIVO: Observar a ocorrência de amebas de vida livre dos gêneros Acanthamoeba e Naegleria em amostras de poeira coletadas em hospitais. MÉTODOS: Foram coletadas 132 amostras de poeira em dois hospitais do município de Presidente Prudente, Säo Paulo. Os locais da coleta foram: Unidade de Terapia Intensiva, Centro Cirúrgico, Isolamento de Moléstias Infecciosas, Berçário, Emergência e Cozinha. As amostras foram semeadas em três meios de cultura: meio de ágar näo nutriente com Escherichia coli, meio de ágar infusäo de soja e microcultivo em meio de Pavlova modificado por Giazzi. As amebas isoladas foram identificadas segundo critérios morfológicos. RESULTADOS: O índice geral de positividade para amebas de vida livre, potencialmente patogênicas, dos gêneros Acanthamoeba e Naegleria, foi de 45,5 por cento, sendo positivas 41,6 por cento das amostras de poeira coletadas no hospital universitário e 50 por cento no hospital estadual. Obtiveram-se 45,5 por cento de positividade do gênero Acanthamoeba e 3,8 por cento para amebas do gênero Naegleria. CONCLUSOES: As amebas de vida livre, potencialmente patogênicas, estavam presentes em todos os ambientes estudados dos dois hospitais, sendo que as espécies do gênero Acanthamoeba foram as isoladas com maior freqüência

In vitro effect of antifungal drugs on pathogenic Naegleria spp.

An ameba of the genus Naegleria causing fatal meningoencephalitis in human subjects was investigated for its sensitivity to antifungal drugs: amphotericin B, ketoconazole, fluconazole and itraconazole. The efficacy of these antifungal drugs for pathogenic Naegleria spp was investigated in three strains isolated from patients who had died of primary amebic meningoencephalitis infection at Siriraj Hospital (1986), Ramathibodi Hospital (1987) and Chachoengsao Hospital (1987). All of the isolates were maintained in axenic culture in the Department of Parasitology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Thailand. The sensitivities of the antifungal drugs (MIC50) were: amphotericin B (0.05-0.5 microg/ml), ketoconazole (0.125 microg/ml), fluconazole (0.5-2.0 mg/ml), and itraconazole (10 mg/ml) (p < 0.05). It is important to explain that ketoconazole is slightly more effective than amphotericin B because its action is directed of the permeability of the amebic membrane. The amebae were more resistant ot fluconazole and itraconazole due to the action of the cytochrome P450 multienzyme (in the case of fluconazole) and the direct effect on heme-iron, blocking cytochrome P450-dependent chitin synthesis (in the case of itraconzole). We conclude that amphotericin B and ketoconazole remain the main drugs with proven activity against pathogenic Naegleria spp.