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)

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.

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.

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.

Distribution of pathogenic Naegleria spp in Thailand.

Research concerning the distribution, isolation, viability, ultrastructure, morphology and immunogenicity of Naegleria fowleri has been increasing in Thailand during 1988-2000. The distribution of the organism was carried out from 1985 to 1987 in Si Sa Ket and Ubon Rachathani Provinces, after the first fatal case was reported in Si Sa Ket. Since then in a 1998 survey of N. fowleri in stagnant water around industrial areas was carried out in Pathum Thani, Samut Prakan and Lopburi provinces. The results showed that 10% of pathogenic Naegleria belonged to species fowleri as characterized by morphology and the occurrence of pathogenesis in mice after nasal inoculation. In the same year, Nacapunchai et al (1999) determined the prevalence of amebae in aquatic habitat of human environments in five parts of Thailand during the summer. Fourteen percent of free living Naegleria spp were found in both soil and water resources. Recent studies of the ultrastructure, factors affecting the viability and SDS-PAGE electrophoretic patterns of 3 Thai strains of pathogenic Naegleria spp indicated their similarities in morphological characteristics of pathogenic reference control, Naegleria fowleri CDC VO 3081. Additional study using a genetic approach to species criteria using allozyme electrophoresis had been conducted.

Amebas patógenas de vida libre / Pathogenic free living amebas

Las pequeñas amebas de vida libre están ampliamente distribuidas en todo el mundo en continuo contacto con el hombre y animales; sus formas quísticas son capaces de sobrevivir en el suelo, aire y agua. Las infecciones causadas por las mismas han tomado en los últimos treinta años notable importancia médica ya que muchos casos fatales no fueron diagnosticados clínicamente ni por el laboratorio, debido al desconocimiento de la potencial capacidad patogénica de estas amebas. Hoy se sabe que la meningoencefalitis amebiana primaria (MAP) causada por Naegleria fowleri y la encefalitits amebiana granulomatosa (EAG) originada por especies de Acanthamoeba spp se han incrementado en el mundo tanto en sujetos sanos como en inmunocomprometidos, incluyendo muchos individuos con SIDA. El grupo más reciente de infecciones causadas por especies del género Acanthamoeba es la queratitis amebiana relacionada principalmente con la falta de cuidado en el mantenimiento de las lentes de contacto. La terapia de la queratitis es problemática debido a la presencia de quistes en los tejidos, y aunque se han informado algunas curas de pacientes, la terapéutica médica aún constituye un capítulo no resuelto

Isolation and identification of free living amoebae from some water sources in Alexandria

Samples from different water sources of Alexandria, and from nasal passages of 500 healthy children inhabiting areas nearby these sources were examined for the presence of free-living amoebae. These samples were cultured on 1.5% non nutrient agar streaked with bacteria. Amoebae were isolated and identified by means of their morphological characters and ability to produce flagellated forms. Characteristics of the cystic stages and pattern of excystation also aided in the identification of the various species. Nine species, Naegleria gruberi, N. fowleri, Acanthamoeba rhysodes, A. glebae, A. culbertsoni, A. astronysis, A. palestinensis, V. avara and V. inornata were isolated from the water of canals and drains. N. gruberi and A. rhysodes were found in the nasal passages of six healthy children living near the contaminated canals. No amoebae were encountered in the drinking water, swimming pools, sea and lake water included in this study

Free-living amoebae in Egypt: 1-naegleria gruberi and naegleria fowleri

Two Naegleria species were isolated and identified from various water sources in Lower and Upper Egypt. Identification was based on the morphology, nuclear division and the excitation and flagellation tests. The trophic, cystic and flagellate forms of N. gruberi are larger than those of N. fowleri and the cyst of the former species has one or more pores, while that of the latter species has no pores and has an outer gelatinous layer. The size and the morphological characteristics of these two free-living amoebae from Egypt were in complete agreement with those previously described for amoebae of the same genus and species endemic to other geographical areas

Characterization of potentially pathogenic free-living amoebae in sewage samples of Calcutta, India

1. It is widely accepted that foul or polluted environments are the principal sources of potentially pathogenic species of free-living amoebae. The present paper is the first report of occurrence of potentially pathogenic free-living amoebae in sewage sample of Calcutta, India. 2. We describe the occurrence, isolation, specific identification and comaprative mouse pathogenicity test of two pathogenic amoebae, viz., Naegleria fowleri (N. aerobia) carter, 1970, causing human meningoencephalitis and Acanthamoeba castellanii Douglas, 1930, causing granulomatous amoebic encephalitis, and one non-pathogenic amoeba, viz., A astronyxis Ray and Hayes, 1959, in sewage samples of Calcuta, India. 3. The existence of both pathogenic and non-pathogenic amoebae living side by side is of considerable epidemiological relevance

Nasal carriage of Naegleria fowleri and its environmental occurrence in Borno State, Nigeria.

The presence of free living amoebae was investigated in the nasal passages of 50 healthy children and in environmental sources in Maiduguri, Borno State of Nigeria. Three of the children yielded positive cultures of Naegleria fowleri. All the five water samples and two of the nine soil samples examined from different localities were positive for N. fowleri. The isolates proved pathogenic for laboratory mice causing a fatal meningoencephalitis. The epidemiological significance of the findings is discussed.

Primary amoebic meningoencephalitis in Thailand: report of a case and review literatures.

Primary amoebic meningoencephalitis (PAM) which is caused by free-living amoeba, Naegleria fowleri, is a rare disease. We report the fifth case in Thailand in order to add more information. The patient was a previously healthy 4 1/2-year-old girl from Nakhon Pathom province. For several weeks before this illness she had swum in a water supply canal. She developed high fever with change in consciousness. Her cerebrospinal fluid contained numerous Naegleria fowleri which grew in culture media and mice inoculation. She did not respond to treatment with intravenous and intraventricular amphotericin B, and oral rifampicin. She died on the fifth day of illness. Water sample from the canal also grew N. fowleri. All five reported cases in Thailand were reviewed. It was found that none of them had been exposed to a common source. Four of the five cases were male, and four cases occurred during the summer months, March to May. These findings agree with worldwide information.