Adhesion of Acanthamoeba on Scleral Contact Lenses According to Lens Shape.

Purpose: To investigate the adhesion of Acanthamoeba to scleral contact lens (ScCL) surface according to lens shape. Methods: Two strains of A. polyphaga (CDC:V062 and ATCC 30461) and one clinical Acanthamoeba isolate, were inoculated onto five contact lens (CL): one first-generation silicone hydrogel (SHCL; lotrafilcon B; adhesion control) containing plasma surface treatment; two ScCL (fluorosilicone acrylate) one containing surface treatment composed of plasma and the other containing plasma with Hydra-PEG, and two CL designed with a flat shape having the same material and surface treatments of the ScCL. Trophozoites that adhered to the lens's surfaces were counted by inverted optical light microscopy. Possible alterations of the lens surface that could predispose amoeba adhesion and Acanthamoeba attached to these lens surfaces were evaluated by scanning electron microscopy (SEM). Results: All strains revealed greater adhesion to the ScCL when compared with the flat lenses (P < 0.001). The clinical isolate and the ATCC 30461 had a higher adhesion (P < 0.001) when compared with the CDC:V062. A rough texture was observed on the surface of the lenses that have been examined by SEM. Also, SEM revealed that the isolates had a rounded appearance on the surface of the ScCL in contrast with an elongated appearance on the surface of the silicone hydrogel. Conclusions: The findings revealed that the curved shape of the ScCL favors amoeba adhesion.

A new continuous-flow solar water disinfection system inactivating cysts of Acanthamoeba castellanii, and bacteria.

Solar water disinfection (SODIS) is an effective and inexpensive microbiological water treatment technique, applicable to communities lacking access to safely managed drinking water services, however, the lower volume of treated water per day (< 2.5 L per batch) is a limitation for the conventional SODIS process. To overcome this limitation, a continuous-flow solar water disinfection system was developed and tested for inactivation of Acanthamoeba castellanii cysts and Escherichia coli, Salmonella Typhimurium, Enterococcus faecalis, and Pseudomonas aeruginosa. The system consisted of a solar heater composed of a cylindrical-parabolic concentrator and a UV irradiator formed by a fresnel-type flat concentrator combined with a cylindrical-parabolic concentrator. Deionized water with low or high turbidity (< 1 or 50 nephelometric turbidity unit (NTU) where previously contaminated by 108 Cysts/L or 105-106 CFU/mL of each of four bacterial species. Then was pumped from the heating tank flowing through the heater and through the UV irradiator, then returning to the heating tank, until reaching 45, 55, 60 or 70 °C. The water was kept at the desired temperature, flowing through the UV irradiator for 0.5 and 10 min. Trophozoites were not recovered from cysts (during 20 days of incubation) when water with < 1 NTU was exposed to UV and 60 °C for 0.5 min. In water with 50 NTU, the same result was obtained after 10 min. In water with < 1 NTU, the inactivation of all bacteria was achieved when the water with < 1 NTU was exposed to 55 °C and UV for 0.5 min; in water, with 50 NTU the same result was achieved by exposure to 60 °C and UV for 0.5 min. The prototype processes 1 L of water every 90s. The system is effective and has the potential to be applied as an alternative to the large-scale public drinking water supply.

Recovery of an Acanthamoeba strain with two group I introns in the nuclear 18S rRNA gene.

Nuclear group I introns are parasitic mobile genetic elements occurring in the ribosomal RNA genes of a large variety of microbial eukaryotes. In Acanthamoeba, group I introns were found occurring in the 18S rDNA at four distinct insertion sites. Introns are present as single elements in various strains belonging to four genotypes, T3 (A. griffini), T4 (A. castellanii complex), T5 (A. lenticulata) and T15 (A. jacobsi). While multiple introns can frequently be found in the rDNA of several algae, fungi and slime moulds, they are usually rare and present as single elements in amoebae. We reported herein the characterization of an A. lenticulata strain containing two introns in its 18S rDNA. They are located to already known sites and show basal relationships with respective homologous introns present in the other T5 strains. This is the first and unique reported case of multiple nuclear introns in Acanthamoeba.

Box Behnken design of siRNA-loaded liposomes for the treatment of a murine model of ocular keratitis caused by Acanthamoeba.

Acanthamoeba keratitis is an ophthalmic disease with no specific treatment that specially affects contact lens users. The silencing of serine phosphatase (SP) and glycogen phosphorylase (GP) proteins produced by Acanthamoeba has been shown to significantly reduce the cytopathic effect, although no vehicle was proposed yet to deliver the siRNA sequences to the trophozoites. In this study, PEGylated cationic liposomes were proposed and optimized using Box-Behnken design. The influence of DOTAP:DOPE ratio, DSPE-PEG concentration, and siRNA/DOTAP charge ratio were evaluated over both biological response and physicochemical properties of liposomes. The ratio of DOTAP:DOPE had an effect in the trophozoite activity whereas the charge ratio influenced both size and protease activity. The predicted values were very close to the observed values, yielding a formulation with good activity and toxicity profile, which was used in the following experiments. A murine model of ocular keratitis was treated with siGP + siSP-loaded liposomes, as well as their respective controls, and combined treatment of liposomes and chlorhexidine. After 15 days of eight daily administrations, the liposomal complex combined with chlorhexidine was the only treatment able to reverse the more severe lesions associated with keratitis. There was 60% complete regression in corneal damage, with histological sections demonstrating the presence of an integral epithelium, without lymphocytic infiltrate. The set of results demonstrate the efficacy of a combined therapy based on siRNA with classical drugs for a better prognosis of keratitis caused by Acanthamoeba.

Modulation of Zinc Homeostasis in Acanthamoeba castellanii as a Possible Antifungal Strategy against Cryptococcus gattii.

Cryptococcus gattii is a basidiomycetous yeast that can be found in the environment and is one of the agents of cryptococcosis, a life-threatening disease. During its life cycle, cryptococcal cells take hold inside environmental predators such as amoebae. Despite their evolutionary distance, macrophages and amoebae share conserved similar steps of phagocytosis and microbial killing. To evaluate whether amoebae also share other antifungal strategies developed by macrophages, we investigated nutritional immunity against cryptococcal cells. We focused on zinc homeostasis modulation in Acanthamoeba castellanii infected with C. gattii. The intracellular proliferation rate (IPR) in amoebae was determined using C. gattii R265 and mutants for the ZIP1 gene, which displays defects of growth in zinc-limiting conditions. We detected a reduced IPR in cells lacking the ZIP1 gene compared to wild-type strains, suggesting that amoebae produce a low zinc environment to engulfed cells. Furthermore, flow cytometry analysis employing the zinc probe Zinpyr-1 confirmed the reduced concentration of zinc in cryptococcal-infected amoebae. qRT-PCR analysis of zinc transporter-coding genes suggests that zinc export by members of the ZnT family would be involved in the reduced intracellular zinc concentration. These results indicate that amoebae may use nutritional immunity to reduce fungal cell proliferation by reducing zinc availability for the pathogen.

Occurrence of Infected Free-Living Amoebae in Cooling Towers of Southern Brazil.

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.

siRNA-loaded liposomes: Inhibition of encystment of Acanthamoeba and toxicity on the eye surface.

Current treatments for Acanthamoeba keratitis are unspecific. Because of the presence of the resilient cyst form of the parasite, the infection is persistent. Silencing the key protein of cyst formation, glycogen phosphorylase, has shown potential for reducing encystment processes of the Acanthamoeba trophozoite. However, a suitable carrier to protect and deliver siRNA sequences is still needed. DOTAP: DOPE:DSPE-PEG liposomes were prepared by three different techniques and used to associate a therapeutic siRNA sequence. Liposomes prepared by film hydration followed by membrane extrusion were considered the most adequate ones with average size of 250 nm and zeta potential of +45 mV, being able to associate siRNA for at least 24 hr in culture medium. siRNA-liposomes could inhibit up to 66% of the encystment process. Cell viability studies demonstrated MTT reduction capacity higher than 80% after 3 hr incubation with this formulation. After 24 hr of incubation, LDH activity ranged for both the formulations from around 4% to 40%. In vivo tolerance studies in mice showed no macroscopic alteration in the eye structures up to 24 hr after eight administrations during 1 day. Histological studies showed regular tissue architecture without any morphological alteration. Overall, these results suggest that the formulations developed are a promising new strategy for the treatment of ocular keratitis caused by Acanthamoeba spp.

Detection and quantification of human adenovirus genomes in Acanthamoeba isolated from swimming pools.

Acanthamoeba is the most common free-living environmental amoeba, it may serve as an important vehicle for various microorganisms living in the same environment, such as viruses, being pathogenic to humans. This study aimed to detect and quantify human adenoviruses (HAdV) in Acanthamoebas isolated from water samples collected from swimming pools in the city of Porto Alegre, Southern Brazil. Free-living amoebae of the genus Acanthamoeba were isolated from water samples, and isolates (n=16) were used to investigate the occurrence of HAdVs. HAdV detection was performed by quantitative real-time polymerase chain reaction (qPCR). HAdVs were detected in 62.5% (10/16) of Acanthamoeba isolates, ranging from 3.24x103 to 5.14x105 DNA copies per milliliter of isolate. HAdV viral loads found in this study are not negligible, especially because HAdV infections are associated with several human diseases, including gastroenteritis, respiratory distress, and ocular diseases. These findings reinforce the concept that Acanthamoeba may act as a reservoir and promote HAdV transmission through water.

Acanthamoeba misidentification and multiple labels: redefining genotypes T16, T19, and T20 and proposal for Acanthamoeba micheli sp. nov. (genotype T19).

Acanthamoeba species are ubiquitous amoebae able to cause important infections in humans and other vertebrates. The full/near-full sequences (>2000 bp) of the small subunit ribosomal RNA gene (SSU rDNA or 18S rDNA) are used to cluster Acanthamoeba as genotypes, labeled T1 to T20. Genotype T15 remains an exception, being described only partially on a 1500-bp fragment. Strains are thus usually identified based on their 18S identity matches with reference strains, often using shorter (<500 bp) diagnostic fragments of the gene. Nevertheless, short fragments (<1000 bp) have been used to propose genotypes. This has been criticized, and doubts arise therefore on possible confusion leading to classify distinct partial sequences with a same label(s). We demonstrate herein that several partial sequences misassigned either to T16 or to T4, actually belong to at least two separate and distinct genotypes. We obtained the full 18S rDNA of a strain previously typed as T16 on the basis of a small fragment and demonstrated that it actually belongs to the recently described T19. We propose the name Acanthamoeba micheli sp. nov., for this strain. Furthermore, partial molecular phylogenies were performed to show that several other misassigned T16 partial sequences belong to a new genotype. This latter includes also misassigned T4 partial sequences, only recently available as full sequences and labeled as T20. We thus reassign these partial sequences to the genotype T20. Longer sequences, ideally at least 90 % of the total gene length, should be obtained from strains to ensure reliable diagnostic and phylogenetic results.

Amoebicidal activity of phytosynthesized silver nanoparticles and their in vitro cytotoxicity to human cells.

Acanthamoeba causes infections in humans and other animals and it is important to develop treatment therapies. Jatropha curcas, Jatropha gossypifolia and Euphorbia milii plant extracts synthesized stable silver nanoparticles (AgNPs) that were relatively stable. Amoebicidal activity of J. gossypifolia, J. curcas and E. milii leaf extracts showed little effect on viability of Acanthamoeba castellanii trophozoites. Plant-synthesized AgNPs showed higher amoebicidal activity. AgNPs synthesized by J. gossypifolia extract were able to kill 74-27% of the trophozoites at concentrations of 25-1.56 µg mL(-1) . AgNPs were nontoxic at minimum inhibitory concentration with peripheral blood mononuclear cells. These results suggest biologically synthesized nanoparticles as an alternative candidate for treatment of Acanthamoeba infections.

Isolation and genotyping of free-living environmental isolates of Acanthamoeba spp. from bromeliads in Southern Brazil.

Species of Acanthamoeba are frequently isolated from distinct environmental sources such as water, soil, dust and air. They are responsible to cause infections and disease in humans and animals. In addition, Acanthamoeba sp. are considered an important reservoir of bacteria, virus and fungi, which act as "Trojan horses" to protect these microorganisms of harsh environmental conditions. In this study, nine Acanthamoeba isolates from bromeliads phylloplane were identified based on the morphology of cyst and trophozoite forms. The genotype level was accessed by the sequence analysis of Acanthamoeba small-subunit rRNA gene. Genotypic characterization grouped five isolates in the genotype T2/T6, three in the T4 genotype and one in the genotype T16. The results obtained indicate that the genotype T2/T6 is common on phylloplane. To predict the pathogenic potential of the Acanthamoeba isolates, thermo and osmotolerance assays were employed, although all isolates were capable of surviving at temperatures of 37°C, other tests will be conducted in the future to determine the potential pathogenic of the isolates. Altogether, our results revealed the importance of the presence of Acanthamoeba associated with bromeliads in Rio Grande do Sul, Brazil, and the necessity for further studies to determine the environmental distribution and the role of these species.

Acanthamoeba T3, T4 and T5 in swimming-pool waters from Southern Brazil.

Species of Acanthamoeba, known to cause keratitis (AK) and granulomatous encephalitis in humans are frequently isolated from a variety of water sources. In this study, 13 Acanthamoeba isolates from swimming pools were classified at the genotype level based on the sequence analysis of the Acanthamoeba small-subunit rRNA gene. Nine of the 13 isolates were genotype T5, three were genotype T4, and one was T3. Several genotypes have been reported worldwide as causative agents of AK, including genotypes T3, T4, and T5. The present study indicates that genotype T5 is a common contaminant in swimming-pool water.

Microbiological quality of minimally processed vegetables sold in Porto Alegre, Brazil

Minimally processed vegetables go through various steps during their preparation, with many modifications to their natural structure. However, they must maintain the same quality as the fresh produce. The aim of the present study was to quantify mesophilic and psychrotrophic microorganisms and total and faecal coliforms, and to assess the presence of Escherichia coli, parasites, and dirt material in ready-to-eat minimally processed vegetables. Fifty-six samples of minimally processed vegetables were analysed for the presence of mesophilic and psychrotrophic microorganisms by the plate-count method. Monthly means ranged from 4.7x10(5) to 1.6x10(8) CFU/g and from 7.9x10(6) to 2.7x10(8) CFU/g, respectively for mesophilic and psychrotrophic microorganisms. Coliforms were analysed by the multiple-tube method; total coliforms ranged from <3 to ³ 2.4x10(4) MPN/g and faecal coliforms from <3 to 1.1x10(4) MPN/g. Escherichia coli was detected in eight samples. Out of 52 samples, eight (15.3 percent) contained oocysts of Eimeria spp.. Dirt matter, such as insect body parts and young mites, was also found. Contamination of faecal origin was observed in these samples, suggesting that either the sanitisation of the product was unsuccessful, or soil or irrigation water could be the source of these microorganisms.

Hortaliças minimamente processadas passam por várias etapas durante seu processamento, no qual ocorrem várias modificações de sua estrutura natural, todavia elas devem manter a mesma qualidade do produto não processado. O objetivo deste estudo foi quantificar microrganismos mesófilos e psicrótróficos, coliformes totais e fecais e verificar a presença de E. coli, parasitas e sujidades em hortaliças minimamente processadas prontas para consumo. Foram analisadas 56 amostras para mesófilos e psicrotróficos pelo método de contagem em placas, com média mensal 4,7x10(5) a 1,6x10(8) UFC/g e de 7,9x10(6) a 2,7x10(8) UFC/g, respectivamente. Os coliformes foram analisados pela técnica dos tubos múltiplos, onde coliformes totais variaram de <3 a ³ 2,4x10(4) NMP/g e coliformes fecais, de <3 a 1,1x10(4) NMP/g, e E. coli foi observada em oito amostras. De 52 amostras, 8 (15,3 por cento) apresentaram oocistos de Eimeria spp. Sujidades, como fragmentos de insetos e ácaros jovens foram encontrados. Contaminação de origem fecal foi verificada no presente estudo, sugerindo falhas nas etapas do processamento das hortaliças, ou que o solo ou a água de irrigação também poderiam ser fontes de disseminação destes microrganismos.

Prevalência das enteroparasitoses nas vilas periéricas da grnde Porto Alegre, nos assentamentos de trabalhadores rurais e na cidade de Arroio dos Ratos, no Estado do Rio Grande do Sul / Survey of intestinal parasites among school students inhabitants of Porto Alegre Metropolitan area and among rural communities of Rio Grande do Sul State

Realizou-se um trabalho intgrado de pesquisa e extensão comunitária com o objetivo de avaliar a prevalência das enteroparasitoses e das condições sócioeconômicas e sanitárias da população residente em 33 vilas periféricas da região metropolitana de Porto Alegre, em 3 assentamentos de trabalhadores rurais e na cidade de Arroio dos Ratos no Estado do Rio Grande do Sul. Esse estudo foi realizado durante o período de 1965 e 1996 em 17951 pessoas, pertencentes a um grupo etário de zero a 15 anos, todos alunos das escolas públicas e em mais dois grupos etários: um e 16 a 20 anos e outro com mais de 20 anos. As amostras fecais foram examinadas pela técnica d Hoffman, Pons e Janer. Das pessoas examinadas, 6,0% (11.855) estavam infectadas por uma ou mais espécies de parasitos intestinais, dos quais 31% (5.581)apresentaram apenas uma espécie de parasito, enquanto que o poliparasitismo representou 35% das respostas, em um total de 6.277 combinações. O maior percentual geral de infecção obtido para nermatóides e cestóides foi de 43,1% (7.092) para Trichuris trichiura e, entre os protozoários, a Giardia lamblia com 17,1% (2.820). As associações mais frequentes, em infecções concomitantes por helmintos e protzoários foram: Ascaria lumbricoides e T trichiura com ,2% (1.835), T trichiura e G lamblia com 8,5% (537) e Entamoeba coli e G lamblia com 2,0% (124). Os resultados obtidos nesse inquérito levam a sugerir necessidade de proporcionar às pessoas residentes nas vilas periféricas, nos assentamentos de trabalhadores rurais e na cidade de Arroio dos Ratos uma Campanha d educação sanitária e, paralelamente, o desenvolvimento de medidas administrativas, visando saneamento básico.

Amebas e amebose: permanente confusäo / Ameba and amebiasis: continuous confusion

A Entamoeba histolytica é um protozoário parasita sendo o agente causador da amebose. A discrepância entre a prevalência e a morbidade pode ser atribuída a ocorrência de distintas formas genéticas de E. histolytica näo patogênicas e potencialmente patogênicas. Extensivos estudos estäo sendo realizados para estabelecer o mecanismo da patogenicidade desta ameba

Blastocystis hominis e o exame parasitológico das fezes / Blastocystis hominis and the routine stool examination

Blastocystis hominis, um parasito intestinal do homem, foi anteriormente descrito como levedura. Ziertd e colaboradores estudaram e reclassificaram este organismo como um protozoário. O B. hominis no exame parasitológico das fezes deverá ser quantificado (raros, poucos, moderados, muitos). No mínimo três exames das fezes, para a pesquisa de ovos e parasitas (incluindo esfregaços permanentes corados), deveräo ser realizados, para assegurar a ausência de outros protozoários, antes de atribuir os sintomas ao B. hominis. A presença de outros agentes microbianos deverá também ser considerada