Isolamento, caracterização biológica e molecular de acanthamoeba: determinação do potencial patogênico no Distrito Federal

O gênero Acanthamoeba pertence a um grupo de protozoários anfizóicos que podem ou não ser patogênicos. A patogenicidade é determinada por parâmetros morfológicos, físicos, bioquímicos e moleculares. Fatores como osmotolerância e a termotolerância contribuem para a determinação da patogenicidade. A técnica de Reação em Cadeia da Polimerase (PCR) vem apresentando resultados mais promissores para a identificação do gênero. O objetivo deste trabalho foi isolar amebas de vida livre do gênero Acanthamoeba em piscinas públicas do DF e espelhos-d’água localizados no Eixo Monumental, Brasília-DF, bem como determinar o potencial patogênico dos isolados. Foi observado crescimento de AVLs em todas as amostras. O gênero Acanthamoeba foi encontrado em 20 amostras pela cultura e confirmados pela PCR. Desses, 14 apresentaram algum grau de patogenicidade nos testes de osmotolerancia e termotolerância

The first Acanthamoeba keratitis case in the Midwest region of Brazil: diagnosis, genotyping of the parasite and disease outcome

We report an Acanthamoeba keratitis case associated with the use of contact lens in a 28-year-old female from Brasília, Brazil. Samples from corneal scraping and contact lens case were used for culture establishment, PCR amplification, and partial sequencing (fragments of ~400kb) of small subunit rDNA; both culture and PCR were positive. The sequence analyses of the cornea and of isolates from the contact lens case showed similarity with the T4 genotype. To the best of our knowledge, this is the first report of T4 Acanthamoeba keratitis case from the Midwest region of Brazil

Isolation and morphological characterization of free-living amoeba from soil and water (swimming pools) samples in Brasília, Distrito Federal, Brazil

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Ciências da Saúde, 2006.As amebas de vida livre são protozoários unicelulares encontrados tanto em solo úmido quanto em ambientes aquáticos. Desenvolvem-se no meio ambiente em solo, poeira e ar; em locais com água sem movimentação, em piscinas, lagos e represas; em soluções de limpeza de lentes de contato; em esgotos, unidades de ar-condicionado, aquecimento e ventilação. Os gêneros com relevância em Saúde Pública são Naegleria, Acanthamoeba, Balamuthia e, mais recentemente, Sappinia. Considerando o reconhecido impacto em Saúde Pública, foi feito um levantamento em Brasília (Distrito Federal) para determinar a presença em amostras ambientais principalmente dos gêneros Acanthamoeba e Naegleria. Os ambientes de procedência das amostras foram piscinas ao ar livre e solo de jardins. Foram coletadas 34 amostras de piscinas e 15 de solo para tentativas de isolamento dos dois gêneros. Observações a fresco das amostras previamente concentradas e culturas “ïn vitro” foram realizadas. As amostras positivas para o gênero Acanthamoeba foram submetidas à classificação nos grupos I, II e/ou III de Pussard & Pons (1977). Os cistos dos dois gêneros foram medidos ao microscópio óptico de luz incidente calibrado com ocular micrométrica e aumento de 1000 vezes. As formas evolutivas foram documentadas na caracterização fenotípica. De acordo com a presença de maior quantidade de formas evolutivas, 13 isolados foram selecionados dentre as amostras positivas para Acanthamoeba spp e Naegleria spp e nomeados em UnB 1 a UnB 13. Os isolados UnB 1 a UnB 6 foram originados de amostras de piscinas e os isolados UnB 7 a UnB 13, de solo. Todos os isolados exibiram uma alta e eficiente proliferação em meio de cultura não definido, ágar – soja, a 25°C. Foram identificadas fenotipicamente, dentre os isolados, 8 (61%) amostras positivas para Acanthamoeba com morfologia similar aos grupos II e III (baseados no tamanho e morfologia dos cistos) e 8 (61%) para Naegleria. Além dos dois citados acima, o gênero Vannella também foi encontrado em 2 (15%) amostras. As formas evolutivas encontradas foram documentados por fotografia digital. Não foi possível demonstrar a presença de formas flageladas de Naegleria. Concluímos que, conforme a literatura especializada, os gêneros Acanthamoeba e Naegleria são freqüentes na natureza, especialmente nos ambientes pesquisados. A caracterização das espécies e o potencial patogênico das mesmas deverá ser estudado posteriormente para as recomendações de medidas de profilaxia. _________________________________________________________________________________________ ABSTRACTFree-living amoebas are unicellular protozoan parasites found in humid soil and aquatic habitats. They develop in soil, dust, air and can also be isolated from motionless water, swimming pools, lakes, dams, contact lenses cleaning solutions, drains, air-conditioner, warming and ventilation units and intestinal lavage fluid. The genera of importance in Public Health are: Naegleria, Acanthamoeba, Balamuthia and Sappinia (recently discovered). Considering the Public Health impact of Acanthamoeba and Naegleria genera, a research was made to verify their existence in environmental samples (openair swimming pools and gardens) in Brasília, Brazil. Some samples from swimming pools (34) and soil (15) were collected in order to verify the presence of free-living amoebas. Direct observation of the samples previously concentrated and in vitro cultures were made. The Acanthamoeba positive samples were classified into the groups I, II and/or III of Pussard & Pons (1977). In an incident light optical microscope, we measured the cysts of the Acanthamoeba and Naegleria genera (1000x magnification); their evolutive stages were registered and phenotypically characterized. Due to the higher number of evolutive stages in comparison with all the Acanthamoeba and Naegleria positive samples, some isolates were selected (UnB 1 to UnB 13). The isolates named UnB 1 to UnB 6 were collected from swimming pools and the isolates named UnB 7 to UnB 13, from soil. We observed an effective and high proliferation in all the isolates with non-defined soy agar culture medium, incubated at 25°C. From all the samples phenotypically identified, the Acanthamoeba genus positive samples represented 61% (8 samples) and showed similarity to the groups II and III of Pussard & Pons (according to the cysts size and morphology); the Naegleria positive samples represented 61% (8 samples). Moreover, we registered the presence of Vannella spp in 15% of the isolates (2 samples). During the experimental tests, we used digital photography to document the evolutive stages seen. It was not possible to demonstrate the presence of the Naegleria flagellated forms. In accordance with the specialized literature, we conclude that Acanthamoeba and Naegleria genera can be frequently seen in nature, especially in soil and water. The characterization of the species and their pathogenic potential should be studied further to recommend prophylaxis

Advances in isolation and biological and molecular characterization of acanthamoeba spp (Acanthamoebidae) – free-living amoeba : experimental determination of pathogenic potential

Tese(doutorado)—Universidade de Brasília, Faculdade de ciências da saúde, Programa de pós-graduação em ciências da saúde, 2012.Várias espécies do gênero Acanthamoeba podem causar ceratite e encefalite amebiana granulomatosa. A avaliação dos critérios de patogenicidade é de grande importância para analisar o risco de infecção. Nossos objetivos foram determinar o potencial patogênico de isolados ambientais de Acanthamoeba de Brasília, DF e avaliar uma técnica de purificação de cultivos de Acanthamoeba. A análise da sequência do 18S rDNA revelou que: entre os 19 isolados estudados, oitoapresentaram similaridade com o genótipo T5, seis com o genótipo T4 e um com o genótipo T2/T6. Os genótipos dos outros quatro isolados não foram determinados. A cepa de referência Acanthamoeba polyphaga (ATCC 30461) serviu de controle. Além disso, 11 isolados (58%) cresceram a 37°C e oito (42%) cresceram em presença de manitol 1,5 M, parâmetros fisiológicos associados com isolados patogênicos de Acanthamoeba, enquanto quatro isolados de água de piscina e um de ceratite apresentaram elevado potencial patogênico. Nos testes de patogenicidade in vivo, realizados com três isolados e a cepa de referência ATCC30461, o isolado de córnea foi considerado patogênico e dois isolados ambientais,invasivos. Foi possível reduzir a porcentagem de fungos e bactérias em uma placa de cultura da cepa de referência ATCC 30461 e de bactérias em uma placa de cultura de um isolado de solo com a inoculação na cavidade peritoneal de camundongos. Nossos resultados indicam ampla distribuição de isolados de Acanthamoeba spp. similares aos genótipos T4, T5 e T2/T6, classificados como invasivos, em diversos habitats de Brasília, DF, revelando o risco potencial de infecção humana e a necessidade de medidas preventivas. _________________________________________________________________________________________ ABSTRACTSeveral Acanthamoeba species may cause keratitis and granulomatous amebic encephalitis. The evaluation of the criteria of pathogen city is very important to assess the risk of infection. We aimed to determine the pathogenic potential of environ mental isolates of Acanthamoeba from Brasília, DF, Brazil, and assess technique to purify Acanthamoeba cultures. Sequence analysis of 18S rDNArevealed that: among the 19 isolates tested, eight were similar to genotype T5, six to genotype T4, and one to genotype T2/T6. The genotypes of the remaining four isolates were not determined. The reference Acanthamoeba polyphaga strain (ATCC30461) was the control. Moreover, 11 strains (58%) presented growth at 37°C and eight (42%) developed in medium containing 1.5 M mannitol, the physiological parameters associated with pathogenic isolates of Acanthamoeba, whereas fours trains isolated from swimming pool water and one from a patient with keratitisshowed high pathogenic potential. In vivo pathogen city tests, performed with three isolates and the reference strain ATCC 30461, showed that the isolate from a patient with keratitis was considered pathogenic and two environmental isolates, invasive. It was possible to reduce the percentage of fungi and bacteria in a culture plate of the reference strain ATCC 30461 and bacteria in a culture plate of one soil isolate byinoculating them into the peritoneal cavity of mice. Our results indicate a wide distribution of Acanthamoeba spp. isolates similar to genotypes T4, T5, and T2/T6, classified as invasive, isolated from several habitats of Brasília, DF, revealing the potential risk of human infection and the need for preventive measures

A method for microbial decontamination of Acanthamoeba cultures using the peritoneal cavity of mice

Objective: To evaluate whether the inoculation of contaminated cultures in the peritoneal cavity of mice could implement decontamination of Acanthamoeba cultures. Methods: Suspensions of Acanthamoeba, Acanthamoeba polyphaga ATCC 30461, or Acanthamoeba spp. isolated from soil (UnB13 strain) were inoculated in the peritoneal cavity of Swiss mice (n = 24). After 1, 6, 12, or 24 h of exposure the peritoneal cavity was washed and assessed for the presence of bacteria, fungi, and Acanthamoeba. Results: After 1 h of intraperitoneal inoculation at least 97% of the bacteria and 96% of the fungi (P < 0.05) and 99% of the bacteria (P < 0.05) were successfully eliminated from the ATCC 30461 strain and from the soil isolate UnB13 strain, respectively. This method also allowed the recovery of most trophozoites and cysts from both Acanthamoeba cultures at the end of 24 h. Conclusions: Our data demonstrated that this technique has great potential for decontamination of Acanthamoeba cultures in a short period of time

NEOTROPICAL XENARTHRANS: a data set of occurrence of xenarthran species in the Neotropics

Xenarthrans—anteaters, sloths, and armadillos—have essential functions for ecosystem maintenance, such as insect control and nutrient cycling, playing key roles as ecosystem engineers. Because of habitat loss and fragmentation, hunting pressure, and conflicts with domestic dogs, these species have been threatened locally, regionally, or even across their full distribution ranges. The Neotropics harbor 21 species of armadillos, 10 anteaters, and 6 sloths. Our data set includes the families Chlamyphoridae (13), Dasypodidae (7), Myrmecophagidae (3), Bradypodidae (4), and Megalonychidae (2). We have no occurrence data on Dasypus pilosus (Dasypodidae). Regarding Cyclopedidae, until recently, only one species was recognized, but new genetic studies have revealed that the group is represented by seven species. In this data paper, we compiled a total of 42,528 records of 31 species, represented by occurrence and quantitative data, totaling 24,847 unique georeferenced records. The geographic range is from the southern United States, Mexico, and Caribbean countries at the northern portion of the Neotropics, to the austral distribution in Argentina, Paraguay, Chile, and Uruguay. Regarding anteaters, Myrmecophaga tridactyla has the most records (n = 5,941), and Cyclopes sp. have the fewest (n = 240). The armadillo species with the most data is Dasypus novemcinctus (n = 11,588), and the fewest data are recorded for Calyptophractus retusus (n = 33). With regard to sloth species, Bradypus variegatus has the most records (n = 962), and Bradypus pygmaeus has the fewest (n = 12). Our main objective with Neotropical Xenarthrans is to make occurrence and quantitative data available to facilitate more ecological research, particularly if we integrate the xenarthran data with other data sets of Neotropical Series that will become available very soon (i.e., Neotropical Carnivores, Neotropical Invasive Mammals, and Neotropical Hunters and Dogs). Therefore, studies on trophic cascades, hunting pressure, habitat loss, fragmentation effects, species invasion, and climate change effects will be possible with the Neotropical Xenarthrans data set. Please cite this data paper when using its data in publications. We also request that researchers and teachers inform us of how they are using these data