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RESUMEN Objetivo: Estandarizar una técnica de reacción en cadena de la polimerasa en tiempo real para la detección del parásito e identificar Acanthamoeba en líquidos conservantes de lentes de contacto. Métodos: Se realizó un estudio descriptivo observacional de corte transversal sobre la técnica de reacción en cadena de la polimerasa en tiempo real para la detección de Acanthamoeba, en el Instituto de Investigaciones en Ciencias de la Salud de la ciudad de Asunción, en Paraguay. Se analizaron 110 líquidos conservantes aportados por usuarios sanos de lentes de contacto, mediante reacción en cadena de la polimerasa en tiempo real y cultivo en medio PAGE - SDS. Resultados: Se estandarizó con éxito la técnica de reacción en cadena de la polimerasa en tiempo real con límite de sensibilidad de 1 pg/µL. Se aisló Acanthamoeba a partir de una muestra (1 por ciento) por método de cultivo, mientras que la carga parasitaria en el líquido conservante fue inferior al límite de detección de la reacción en cadena de la polimerasa en tiempo real. El ADN obtenido del cultivo de dicha muestra fue positivo para Acanthamoeba por este método. Conclusión: El sistema estandarizado presenta buena sensibilidad y podrá ser incorporado en los laboratorios que cuentan con acceso a equipos de reacción en cadena de la polimerasa en tiempo real para un diagnóstico rápido y más eficiente en casos de sospechas de queratitis amebiana. Recomendamos el uso combinado de métodos moleculares y cultivo para aumentar la potencia del diagnóstico, sobre todo en muestras donde la carga parasitaria es muy baja(AU)

ABSTRACT Objective: Standardize a real-time polymerase chain reaction technique for detection of the parasite and identify Acanthamoeba in contact lens solutions. Methods: A cross-sectional observational descriptive study was conducted about a real-time polymerase chain reaction technique for detection of Acanthamoeba at the Institute of Health Sciences Research in the city of Asunción, Paraguay. A total 110 solutions were analyzed, which were provided by healthy contact lens users, by real-time polymerase chain reaction and culture in SDS-PAGE medium. Results: Successful standardization was achieved of the real-time polymerase chain reaction technique with a sensitivity limit of 1 pg/µl. Acanthamoeba was isolated from one sample (1 percent) by culture, whereas the parasite load in the contact lens solution was below the detection limit of the real-time polymerase chain reaction technique. The DNA obtained from the culture of that sample was positive for Acanthamoeba by the real-time polymerase chain reaction technique method. Conclusion: The system standardized exhibits good sensitivity and may be incorporated into laboratories with real-time polymerase chain reaction technique equipment for a rapid and more efficient diagnosis of suspected amoebic keratitis. We recommend the combined use of molecular methods and culture to enhance diagnostic power, mainly in samples where the parasite load is very low(AU)

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This study determined the occurrence of potentially pathogenic free-living amoebae (FLA) and bacteria associated with amoebae in air-conditioning cooling towers in southern Brazil. Water samples were collected from 36 cooling systems from air-conditioning in the state of Rio Grande do Sul, Brazil. The organisms were identified using polymerase chain reaction (PCR) and sequencing automated. The results showed that these aquatic environments, with variable temperature, are potential "hot spots" for emerging human pathogens like free-living amoebae and bacteria associated. In total, 92% of the cooling-tower samples analyzed were positive for FLA, and Acanthamoeba was the dominant genus by culture and PCR. Amoebal isolates revealed intracellular bacteria in 39.3% of them and all were confirmed as members of the genus Pseudomonas. The results obtained show the important role of cooling towers as a source of amoebae-associated pathogens.

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The interactions that occur between bacteria and amoebae can give through mutual relations, where both organisms benefit from the association or parasitic in which one organism benefits at the expense of the other. When these organisms share the same environment, it can result in some changes in the growth of organisms, in adaptation patterns, in morphology, development or even in their ability to synthesize proteins and other substances. In this study, the interaction between Acanthamoeba polyphaga and Staphylococcus aureus (MRSA) was evaluated using a co-culture model at different incubation times. The results showed that 89% of amoebic cells remained viable after contact with the bacteria. The bacterial isolate was visualized inside the amoeba through confocal microscopy and fluorescence for up to 216 h of co-cultivation. The lysate of amoebic culture increased the growth of S. aureus (MRSA), and the effect of supernatant of culture inhibited bacterial growth over the incubation times, suggesting that A. polyphaga produced some metabolite, that inhibited the growth of bacteria. Moreover, the encystment of the A. polyphaga was increased by the bacteria presence. The results show that A. polyphaga and S. aureus interaction may have an important influence on survival of both, and specially indicate a possible effect on the metabolics characteristics each other.

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Amoebae of the genus Acanthamoeba occur worldwide and in addition to being pathogens, are important vehicles for microorganisms with clinical and environmental importance. This study aimed to evaluate the profiling of endosymbionts in 12 isolates of Acanthamoeba using V3 region of 16S rDNA denaturing gradient gel electrophoresis (DGGE) and sequencing. The DGGE enabled us to characterize the endosymbionts diversity in isolates of Acanthamoeba, and to identify Paenibacillus sp., an emerging pathogen, as an amoebic endosymbiont. The results of this study demonstrated that Acanthamoeba is capable of transporting a large number of endosymbionts. This is the first study that reports, the presence of Paenibacillus sp. as amebic symbiont.

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Acanthamoeba is a "Trojan horse" of the microbial world. The aim of this study was to identify the presence of Pseudomonas as an amoeba-resistant microorganism in 12 isolates of Acanthamoeba. All isolates showed the genus Pseudomonas spp. as amoeba-resistant microorganisms. Thus, one can see that the Acanthamoeba isolates studied are hosts of Pseudomonas.

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Acanthamoeba is a “Trojan horse” of the microbial world. The aim of this study was to identify the presence of Pseudomonas as an amoeba-resistant microorganism in 12 isolates of Acanthamoeba. All isolates showed the genus Pseudomonas spp. as amoeba-resistant microorganisms. Thus, one can see that the Acanthamoeba isolates studied are hosts of Pseudomonas.

Acanthamoeba é um “Cavalo de Tróia” do mundo microbiano. Este estudo teve como objetivo identificar a presença de Pseudomonas como microrganismo resistente a ameba em 12 isolados de Acanthamoeba. Todos os isolados apresentaram o gênero Pseudomonas spp. como um microrganismo resistente a ameba. Assim, podemos ver que os isolados de Acanthamoeba estudados são hospedeiros de Pseudomonas.

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Os avanços mais recentes da Oftalmologia poderiam ser resumidos no uso dos implantes intra-oculares na cirurgia de catarata, nas lentes de contacto de uso prolongado e na cirurgia refrativa da córnea. Estes procedimentos säo benéficos, mas suas repercussöes sobre a córnea podem gerar processos patológicos. O grande perigo de infecçöes corneanas com o uso de lentes de contacto de uso prolongado é efnatizado, bem como a necessidade de reconhecer novas formas clínicas de úlceras corneanas causadas por microorganismos pouco conhecidos como Acantameba nestes pacientes

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