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Las Amebas de Vida Libre (AVL) son protozoos anfizoicos de amplia distribución en la naturaleza. Cuatro géneros pertenecientes a este grupo han demostrado ser patógenos para el hombre y animales de experimentación: Naegleria, Balamuthia, Acanthamoeba y Sappinia. En particular, a Acanthamoeba se la considera una AVL “ubicuista”, ya que se la ha hallado en todos los ambientes y lugares donde se la ha investigado. El objetivo de esta tesis fue la búsqueda, aislamiento e identificación de AVL aguas. Los sitios de muestreo correspondieron a la Ciudad de Bahía Blanca y a otros sitios geográficos de la Republica Argentina. En Bahía Blanca se analizaron muestras de agua corriente y de tanques de reserva domiciliarios, de aguas recreacionales (piscinas cubiertas) y arroyos. Se tomaron muestras del Rio Sauce Grande en la localidad bonaerense de Sierra de la Ventana. Se muestrearon aguas de consumo y recreación de la Localidad de Sarmiento, provincia del Chubut. Se analizaron muestras de diversas fuentes en distintas provincias de la Republica Argentina. En total se procesaron 274 muestras de aguas de diferentes fuentes y localizaciones. Las muestras fueron clasificadas en aguas tratadas (cloradas u otro tratamiento: tanques, grifos, piscinas, etc) y naturales (de ríos, lagos, canales, arroyos, etc.). Dentro de esta clasificación solo el 30% de las aguas tratadas analizadas fueron positivas para el aislamiento de AVL, mientras que dentro de las aguas naturales se obtuvo un 92,6% de positividad en los cultivos. Del total de estos aislamientos, el 98,7% correspondió al género Acanthamoeba, tanto por clasificación morfológica como por métodos moleculares; mientras que 1,3% restante correspondió a una cepa que denominamos “No A, no N”, sin ubicación genérica determinada y de cuyo análisis ultraestructural se desprendió la existencia de endosimbiontes compatibles con partículas virales. Se analizaron también muestras clínicas procedentes de 4 posibles casos de queratitis amebiana humana, resultando 2 de ellos positivos para AVL, y que correspondieron al género Acanthamoeba. Se investigó la presencia de AVL en líquidos comerciales para lentes de contacto, y en diversas muestras biológicas procedentes de un hospital de Bahía Blanca. En ambos tipos de muestras los aislamientos resultaron negativos. En todas las cepas aisladas se realizaron análisis morfométricos, ultraestructurales y moleculares, tendientes a caracterizar los aislamientos y confirmar su ubicación genérica. Además se realizaron pruebas de resistencia a diferentes desinfectantes. Nuestros resultados refuerzan la adjetivación de “ubicuista” para las AVL del género Acanthamoeba, ya que fueron demostradas en todos la ambientes estudiados. Su amplia distribución, el hallazgo en aguas recreacionales y de consumo y su participación como agentes etiológicos de enfermedad humana, refuerzan la necesidad de implementar medidas de control y prevención, así como su consideración por parte del personal de salud al momento de realizar un diagnostico clínico e instaurar un tratamiento específico. / The free-living amoebae (FLA) are amphizoic protozoa widely distributed in nature. Four genera belonging to this group have proved to be pathogenic to humans and experimental animals: Naegleria, Balamuthia, Acanthamoeba and Sappinia. In particular, Acanthamoeba is considered an FLA "ubiquitous" since it has been found in all environments and places where it has been investigated. The aim of this thesis was the search, isolation and identification of FLA in the waters. Sampling sites were for the city of Bahia Blanca and other geographical sites of Argentina. In Bahia Blanca samples of tap water tanks and household reserves, recreational water (indoor) and streams they were analyzed. Sauce Grande River samples were taken in the locality of Sierra de la Ventana. Drinking water and recreation of the town of Sarmiento, Chubut province were sampled. Samples from different sources in different provinces of Argentina were analyzed. In total 274 samples of water from different sources and locations were processed. The samples were placed in treated water (chlorinated or other treatment tanks, faucets, pools, etc.) and natural (rivers, lakes, canals, streams, etc.). Within this classification, only 30% of treated water tested positive for isolation of FLA, while natural waters within 92.6% positivity in the cultures was obtained. Of all the isolates, 98.7% were Acanthamoeba, both morphological classification by molecular methods; while remaining 1.3% corresponded to a strain called "non-A, non-N" without certain generic location and ultrastructural analysis which came off the existence of endosymbionts compatible with viral particles. Clinical samples from 4 possible human cases of amoebic keratitis were also analyzed, resulting in 2 of them positive for AVL, which corresponded to Acanthamoeba. FLA's presence in commercial liquid for contact lenses, and various biological samples from a hospital in Bahia Blanca was investigated. In both types of samples were negative isolates. Morphometric, ultrastructural and molecular, aimed to characterize the isolates and confirm your generic location analyzes were performed on all isolates. Additionally stress tests were conducted at different disinfectants. Our results reinforce the adjectives of "ubiquitous" for AVL, especially Acanthamoeba, as were demonstrated in all the studied environments. Its wide distribution, the finding in recreational waters and drinkig waters, and their participation as etiologic agents of human disease, reinforcing the need to implement prevention and control measures, as well as consideration by health personnel when making a clinical diagnosis and establish specific treatment.

Il a été bien documenté que les différentes canalisations des unités de soins dentaires contiennent un épais biofilm. Ce biofilm est constitué entre autres de bactéries, mais aussi d’amibes. Certaines amibes ont un potentiel pathogène et peuvent causer des infections graves. Deux cas d’infections amibiennes et possiblement reliées aux unités dentaires ont retenu notre attention et sont à l’origine du présent projet. L’identification morphologique des amibes afin de déterminer si elles présentent un potentiel pathogène ou non est une tâche ardue, même pour les protozoologistes chevronnés. Nous avons donc utilisé la réaction de polymérase en chaîne (PCR) pour identifier les amibes. Des nouvelles amorces ont été élaborées pour détecter les amibes des genres Acanthamoeba ainsi que Naegleria. Des échantillons d’eau et de terre ont été prélevés dans l’environnement, et des échantillons d’eau et de biofilm ont été prélevés dans les unités dentaires. Une partie de chaque échantillon a été mise en culture selon une méthode améliorée pour une identification morphologique, et l’autre partie a été soumise à un PCR direct. Des Acanthamoebae et/ou des Naegleriae ont été détectées dans 100% des échantillons, mais les espèces varient d’un échantillon à l’autre. Des amibes à potentiel pathogènes sont détectables dans les unités dentaires ainsi que dans l’environnement, et celles-ci pourraient représenter un risque pour la santé de certains individus. / It has been well documented that the various tubing of a dental unit are covered with a thick biofilm. This biofilm mostly consists of bacteria, but amoebae can be found within the biofilm as well. Some amoebae are potential pathogens and may cause serious infections. Two cases of amoebic infections that were possibly linked with dental units drew our attention and stimulated our researches. Morphologic identification of amoebae in order to determine their possible pathogenicity requires much expertise, and is even difficult for proficient protozoologists. Therefore, the use of PCR is essential to detect potentially pathogenic amoebae with subjectivity. We elaborated new primers for the detection of Acanthamoeba spp. and Naegleria spp. Samples of water and dirt were taken in the environment, and samples of water and biofilm were taken in dental units. A part of each samples was cultivated for morphological identification, when a second part was utilized for PCR identification. Acanthamoebae and/or Naegleriae were detected in 100% of our samples, but the species varied from one sample to another. Potentially pathogenic amoebae were detected in dental units and in the environment, which could represent a health risk for some individuals.

Organismos do gênero Acanthamoeba são muito pouco investigados no Brasil, não se conhecendo a diversidade genética e o potencial patogênico das espécies prevalentes no país. Este estudo examinou a presença de Acanthamoeba em 118 amostras de solo e água de diversas regiões do Brasil e, como resultado, 38 novas culturas axenicas foram estabelecidas. A partir do sequenciamento da subunidade menor do gene ribossômico foi possível genotipar, avaliar o polimorfismo genético das amostras brasileiras e também inferir relações filogenéticas entre isolados clínicos e de ambiente do Brasil e entre esses e as demais espécies/isolados de Acanthamoeba. Para alguns grupos de isolados brasileiros, o potencial patogênico, inferido por indicadores indiretos de virulência (termo- e osmotolerância, secreção de peptidases e efeito citopático), foi também investigado. / Organisms of the genus Acanthamoeba are poorly investigated in Brazil, not knowing the genetic diversity and pathogenic potential of the species prevalent in the country. This study examined the presence of Acanthamoeba in 118 soil and water samples from different regions of Brazil and as a result, 38 new axenic cultures were established. From the sequencing of small subunit ribosomal gene was possible to genotype, to assess the genetic polymorphism of Brazilian samples and also to infer phylogenetic relationships among clinical and environmental isolates from Brazil and between these and other species/isolates of Acanthamoeba. For some groups of Brazilian isolates, pathogenic potential, inferred by indirect indicators of virulence (thermo-and osmotolerance, secretion of peptidases and CPE), was also investigated.

Organismos do gênero Acanthamoeba são muito pouco investigados no Brasil, não se conhecendo a diversidade genética e o potencial patogênico das espécies prevalentes no país. Este estudo examinou a presença de Acanthamoeba em 118 amostras de solo e água de diversas regiões do Brasil e, como resultado, 38 novas culturas axenicas foram estabelecidas. A partir do sequenciamento da subunidade menor do gene ribossômico foi possível genotipar, avaliar o polimorfismo genético das amostras brasileiras e também inferir relações filogenéticas entre isolados clínicos e de ambiente do Brasil e entre esses e as demais espécies/isolados de Acanthamoeba. Para alguns grupos de isolados brasileiros, o potencial patogênico, inferido por indicadores indiretos de virulência (termo- e osmotolerância, secreção de peptidases e efeito citopático), foi também investigado. / Organisms of the genus Acanthamoeba are poorly investigated in Brazil, not knowing the genetic diversity and pathogenic potential of the species prevalent in the country. This study examined the presence of Acanthamoeba in 118 soil and water samples from different regions of Brazil and as a result, 38 new axenic cultures were established. From the sequencing of small subunit ribosomal gene was possible to genotype, to assess the genetic polymorphism of Brazilian samples and also to infer phylogenetic relationships among clinical and environmental isolates from Brazil and between these and other species/isolates of Acanthamoeba. For some groups of Brazilian isolates, pathogenic potential, inferred by indirect indicators of virulence (thermo-and osmotolerance, secretion of peptidases and CPE), was also investigated.

Neste trabalho foram caracterizadas molecularmente e inferidas as relações filogenéticas de 14 isolados brasileiros de Acanthamoeba, provenientes de casos de ceratite, e 8 isolados da ATCC (4 de ceratite e 4 ambientais). Foram utilizados inicialmente os métodos de RAPD, RFLP de DNA genômico total e RFLP do SSU rDNA. Apesar de revelar a alta variabilidade genética em Acanthamoeba, estes métodos permitiram estabelecer grupos bem definidos de isolados mais similares geneticamente. O seqüenciamento do SSU rDNA permitiu a inferência da filogenia entre os isolados utilizados nesse estudo em relação àqueles presentes na literatura, que estão distribuídos em doze tipos de seqüência deste gene. Dentre os 17 isolados de ceratite presentes em nosso estudo, 16 apresentaram SSU rDNA tipo T4 (anteriormente já fortemente correlacionado à ceratite) e um deles constitui um novo tipo de seqüência. Dois dos 4 isolados ATCC (ambientais) cujas seqüências ainda não haviam sido determinadas também apresentaram novos tipos de SSU rDNA, enquanto outros 2 apresentaram o tipo T4. / In this work we performed the molecular phylogeny and characterization of 22 Acanthamoeba isolates, 14 Brazilian keratitis isolates and 8 from ATCC, 4 keratitis and 4 environmental isolates. In spite of the extensive genetic variability disclosed by RAPD, total genomic DNA RFLP and SSU rDNA RFLP techniques, these methods enabled us to group some isolates in well defined clusters of genetically more related organisms. Sequencing of SSU rDNA allowed inference of the phylogeny of our isolates with those present in the literature, which are distributed through 12 sequence types of this gene. Among the 17 keratitis isolates of our study, 16 presented SSU rDNA of type T4 (previously found to be strongly correlated to keratitis), and one was assigned to a new sequence type. Of the 4 isolates from ATCC whose sequences were previously undetermined, the two environmental isolates also constituted new sequence types, while the two keratitis isolates were assigned to type T4.

Il a été bien documenté que les différentes canalisations des unités de soins dentaires contiennent un épais biofilm. Ce biofilm est constitué entre autres de bactéries, mais aussi d’amibes. Certaines amibes ont un potentiel pathogène et peuvent causer des infections graves. Deux cas d’infections amibiennes et possiblement reliées aux unités dentaires ont retenu notre attention et sont à l’origine du présent projet. L’identification morphologique des amibes afin de déterminer si elles présentent un potentiel pathogène ou non est une tâche ardue, même pour les protozoologistes chevronnés. Nous avons donc utilisé la réaction de polymérase en chaîne (PCR) pour identifier les amibes. Des nouvelles amorces ont été élaborées pour détecter les amibes des genres Acanthamoeba ainsi que Naegleria. Des échantillons d’eau et de terre ont été prélevés dans l’environnement, et des échantillons d’eau et de biofilm ont été prélevés dans les unités dentaires. Une partie de chaque échantillon a été mise en culture selon une méthode améliorée pour une identification morphologique, et l’autre partie a été soumise à un PCR direct. Des Acanthamoebae et/ou des Naegleriae ont été détectées dans 100% des échantillons, mais les espèces varient d’un échantillon à l’autre. Des amibes à potentiel pathogènes sont détectables dans les unités dentaires ainsi que dans l’environnement, et celles-ci pourraient représenter un risque pour la santé de certains individus. / It has been well documented that the various tubing of a dental unit are covered with a thick biofilm. This biofilm mostly consists of bacteria, but amoebae can be found within the biofilm as well. Some amoebae are potential pathogens and may cause serious infections. Two cases of amoebic infections that were possibly linked with dental units drew our attention and stimulated our researches. Morphologic identification of amoebae in order to determine their possible pathogenicity requires much expertise, and is even difficult for proficient protozoologists. Therefore, the use of PCR is essential to detect potentially pathogenic amoebae with subjectivity. We elaborated new primers for the detection of Acanthamoeba spp. and Naegleria spp. Samples of water and dirt were taken in the environment, and samples of water and biofilm were taken in dental units. A part of each samples was cultivated for morphological identification, when a second part was utilized for PCR identification. Acanthamoebae and/or Naegleriae were detected in 100% of our samples, but the species varied from one sample to another. Potentially pathogenic amoebae were detected in dental units and in the environment, which could represent a health risk for some individuals.

Made available in DSpace on 2016-12-23T13:56:14Z (GMT). No. of bitstreams: 1 Cynara Oliveira Possamai.pdf: 3245812 bytes, checksum: 7de0caeadb98d478a083db43020f22f8 (MD5) Previous issue date: 2012-08-28 / O gênero Acanthamoeba compreende protozoários anfizóicos que estão presentes nos mais diversos ambientes, podendo causar no homem doenças graves, como a ceratite amebiana e a encefalite amebiana granulomatosa. Os fatores envolvidos na patogenicidade de Acanthamoeba não são inteiramente conhecidos, por isso, alguns marcadores vêm sendo investigados na tentativa de identificar linhagens capazes de causar infecção. O objetivo deste trabalho foi investigar a ocorrência de Acanthamoeba em amostras clínicas e ambientais, bem como caracterizar os isolados obtidos por parâmetros morfológicos, genotipagem e avaliação do potencial patogênico. Foram coletadas amostras de raspado de córnea de pacientes com suspeita de ceratite amebiana e amostras ambientais provenientes de poeira, solo, piscina, água potável, água de inundação e água do mar da região metropolitana de Vitória-ES. Todas as amostras foram cultivadas em meio ágar soja. Além da cultura, as amostras de raspado de córnea também foram coletadas em salina de Page e submetidas a uma reação de semi-nested PCR. Culturas positivas para Acanthamoeba, identificadas com base na morfologia dos cistos e trofozoítos, foram selecionadas, clonadas e classificadas nos grupos morfológicos I, II ou III. A genotipagem dos isolados foi realizada a partir do sequenciamento parcial do gene 18S rDNA e o potencial patogênico das culturas clonadas foi avaliado por meio de ensaios de termotolerância e osmotolerância. Foram cultivadas em ágar 90 amostras ambientais, 16 de raspado de córnea e nove de lentes de contato (LC). Dessas, 38 (33 ambientais, quatro clínicas e uma de LC) foram positivas para Acanthamoeba, sendo obtidos 28 clones (24 ambientais, três clínicos e um de LC). Dentre eles, 26 apresentaram características morfológicas do grupo II, um do grupo I (solo) e um clone (água potável) não foi classificado de acordo com os parâmetros morfológicos de classificação. Quatro casos de ceratite amebiana foram confirmados somente por diagnóstico molecular. Todos os isolados clínicos, o de LC e a maioria dos isolados ambientais sequenciados foram classificados como pertencentes ao genótipo T4. Dentre os isolados ambientais, dois foram agrupados no genótipo T11 (piscina) e um no T1 (poeira). Todos os isolados clonados submetidos aos ensaios de termotolerância apresentaram crescimento a 28ºC e a 37ºC. Em contrapartida, nenhum isolado cresceu a 42ºC. Nos testes de osmotolerância, todos os isolados se desenvolveram a 0,1M e a 0,5M de manitol e a maioria deles cresceu à concentração de 1,0M. Os resultados deste estudo pioneiro no Espírito Santo confirmam a predominância do grupo morfológico II e do genótipo T4 em isolados clínicos e ambientais de Acanthamoeba e relata pela primeira vez no Brasil o isolamento de Acanthamoeba pertencente ao genótipo T1. Este trabalho demonstra também a presença de isolados potencialmente patogênicos no ambiente, inclusive em amostras de água de inundação e de água do mar, o que pode representar um fator de risco para o desenvolvimento de infecções causadas por Acanthamoeba. Além disso, a metodologia desenvolvida neste estudo poderá ser utilizada para um diagnóstico mais rápido, sensível e específico de ceratite por Acanthamoeba / The genus Acanthamoeba comprises amphizoic protozoa with a wide environment distribution. They can cause serious diseases in humans, such as amoebic keratitis and granulomatous amoebic encephalitis. Thus, the factors involved in the pathogenicity of Acanthamoeba are being investigated as major interests to identify strains able to cause infection. The aim of this study was to investigate the occurrence of Acanthamoeba both in clinical and environmental samples, characterize the isolates by morphological parameters, genotyping and also evaluate the pathogenic potential. Clinical samples were collected from patients with a suspicious diagnosis of amoebic keratitis throughout corneal scrapings. Environmental samples were collected from dust, soil, swimming pool, tap, sea, and flood waters in Vitoria metropolitan regions, Espirito Santo State, Brazil. All samples were cultured on soy agar. Samples from corneal scrapings were also collected in Page saline and subjected to a reaction of semi-nested PCR. Positive cultures for Acanthamoeba, previously identified based on the cysts and trophozoites morphology, were selected, cloned and classified into morphological groups I, II or III. Genotyping of isolates was performed by partially sequencing the 18S rDNA gene while the pathogenic potential of cloned cultures was assessed by thermo and osmotolerance assays. From all samples cultured on agar, 90 were from environmental sources, 16 from corneal scrapings and nine from contact lenses (CL). Of these, 38 (33 from environmental samples, four from clinical samples and one from CL sample) were positive for Acanthamoeba. Among the 38 positive isolates, 28 were successfully cloned (24 from environmental isolates, three from clinical isolates and one from CL isolate). Twenty six cloned samples showed morphological characteristics of group II, one of group I (soil) and one (tap water) could not be classified according to morphological parameters. Four cases of amoebic keratitis could only be confirmed by molecular diagnosis. All clinical, CL and most of the environmental isolates sequenced were classified as T4 genotype. Among the environmental isolates, two were grouped in genotype T11 (pool) and one in T1 (dust). All cloned isolates subjected to the thermotolerance assay grew at 28 ºC and 37 ºC. The same result was observed in osmotolerance tests at 0.1M and 0.5M mannitol. Neverthless, while most of the cloned isolates were able to grow at 1.0M mannitol, none of the isolates were able to grow at 42 ºC. The present study confirms the predominance of morphological group II and genotype T4 in clinical and environmental isolates of Acanthamoeba in Espirito Santo State and first time reports the T1 genotype isolation of Acanthamoeba in Brazil. This work also demonstrates the presence of potentially pathogenic isolates at the environment, including samples of flood and sea waters, which may represent a risk factor for the development of infections caused by Acanthamoeba. Furthermore, the methodology used in this study could be used for a fast, sensitive and specific diagnosis of Acanthamoeba keratitis

Neste trabalho foram caracterizadas molecularmente e inferidas as relações filogenéticas de 14 isolados brasileiros de Acanthamoeba, provenientes de casos de ceratite, e 8 isolados da ATCC (4 de ceratite e 4 ambientais). Foram utilizados inicialmente os métodos de RAPD, RFLP de DNA genômico total e RFLP do SSU rDNA. Apesar de revelar a alta variabilidade genética em Acanthamoeba, estes métodos permitiram estabelecer grupos bem definidos de isolados mais similares geneticamente. O seqüenciamento do SSU rDNA permitiu a inferência da filogenia entre os isolados utilizados nesse estudo em relação àqueles presentes na literatura, que estão distribuídos em doze tipos de seqüência deste gene. Dentre os 17 isolados de ceratite presentes em nosso estudo, 16 apresentaram SSU rDNA tipo T4 (anteriormente já fortemente correlacionado à ceratite) e um deles constitui um novo tipo de seqüência. Dois dos 4 isolados ATCC (ambientais) cujas seqüências ainda não haviam sido determinadas também apresentaram novos tipos de SSU rDNA, enquanto outros 2 apresentaram o tipo T4. / In this work we performed the molecular phylogeny and characterization of 22 Acanthamoeba isolates, 14 Brazilian keratitis isolates and 8 from ATCC, 4 keratitis and 4 environmental isolates. In spite of the extensive genetic variability disclosed by RAPD, total genomic DNA RFLP and SSU rDNA RFLP techniques, these methods enabled us to group some isolates in well defined clusters of genetically more related organisms. Sequencing of SSU rDNA allowed inference of the phylogeny of our isolates with those present in the literature, which are distributed through 12 sequence types of this gene. Among the 17 keratitis isolates of our study, 16 presented SSU rDNA of type T4 (previously found to be strongly correlated to keratitis), and one was assigned to a new sequence type. Of the 4 isolates from ATCC whose sequences were previously undetermined, the two environmental isolates also constituted new sequence types, while the two keratitis isolates were assigned to type T4.