Viability of Acanthamoeba cysts in ophthalmic solutions.

Acanthamoeba keratitis is a chronic infection of the human cornea. Many people who have this infection wear soft contact lenses. Usually lens wearers clean and maintain their lenses with various ophthalmic solutions including homemade saline. Recently it has been shown that homemade saline solutions play a role in lens contamination and thus in Acanthamoeba keratitis. We therefore evaluated the viability of cysts of three species of Acanthamoeba by exposing them for various time periods to saline, cleaning, and disinfectant solutions generally used to care for these lenses. We found that the viability of the cysts in saline solutions ranged from a minimum of 14 days to 90 days of exposure. In cleaning solutions, the survival times ranged from a minimum of 1 day to 90 days of exposure. Disinfectants, as expected, were the most effective of all tested solutions in killing Acanthamoeba cysts. The survival times ranged from 6 h to 14 days. None of these products were effective in destroying Acanthamoeba cysts in less than 6 h of exposure, which exceeds the suggested time that any given solution should be used for lens care.     

Moist-heat disinfection of pathogenic Acanthamoeba cysts

The effect of moist-heat in the disinfection of pathogenic Acanthamoeba cysts was investigated. Temperatures of 56°C or 60°C were not effective in killing cysts of A. polyphaga even after a contact time of 60 min, A 4 log reduction in viability was achieved within 15 min at 65°C and 2 min at 70°C giving decimal reduction rates (D-values) of 3·75 min and 30 s respectively. The ability of a commercial moist-heat contact lens disinfection unit to kill 1 times 10⁵ cysts of Acanthamoeba isolated from contact lens storage cases was also shown. Holding temperatures inside the cases of 65°C for 5·5 min and 70°C (the maximum temperature obtained) for 1 min were recorded during the disinfection cycle.     

Photocatalytic disinfection of Giardia intestinalis and Acanthamoeba castellani cysts in water

In this study, disinfection of water containing Giardia intestinalis and Acanthamoeba castellani cysts with TiO2 and modified catalyst silver loaded TiO2 (Ag-TiO2) was investigated. Destruction of the parasites was evaluated after UV illumination of the suspension consisting 5 x 10(8)-13.5 x 10(8)cysts/mL in the presence of 2g/L neat or modified TiO2 at neutral pH. In the initial stage, the solid photocatalyst particles penetrated the cyst wall and then oxidant species produced by TiO2/UV destroyed both cell wall and intracellular structure. In the case of G. intestinalis inactivation (disinfection) performance of TiO2/UV system reached 52.5% only after 25 min illumination and total parasite disinfection was achieved after 30 min illumination. However, silver loaded TiO2 seemed to be more effective as this loading provided better catalytic action as well as additional antimicrobial properties. Cell viability tests showed that parasite cysts, their walls in particular, were irreversibly damaged and cysts did not re-grow. Nevertheless the studied system seemed to be ineffective for the inactivation of A. castellani. Inactivation percentages of TiO2/UV and Ag-TiO2/UV systems were far lower than that of UV alone, being 50.1% and 46.1%, respectively.     

The interaction of Acanthamoeba castellanii cysts with macrophages and neutrophils

Acanthamoeba castellanii, a free-living amoeba, causes a sight-threatening form of keratitis. Even after extensive therapies, corneal damage can be severe, often requiring corneal transplantation to restore vision. However, A. castellanii cysts are not eliminated from the conjunctiva and stroma of humans and can excyst, resulting in infection of the corneal transplant. The aim of this study was to determine whether elements of the innate immune apparatus, neutrophils and macrophages, were capable of detecting and eliminating A. castellanii cysts and to examine the mechanism by which they kill the cysts. Results show that neither innate immune cell is attracted chemotactically to intact cysts, yet both were attracted to lysed cysts. Both macrophages and neutrophils were capable of killing significant numbers of cysts, yet neutrophils were 3-fold more efficient than macrophages. Activation of macrophages with lipopolysaccharide and interferon-gamma did not increase their cytolytic ability. Conditioned medium isolated from macrophages did not lyse the cysts; however, prevention of phagocytosis by cytochalasin D inhibited 100% of macrophage-mediated killing of the cysts. Conditioned medium from neutrophils did kill significant numbers of the cysts, and this killing was blocked by quercetin, a potent inhibitor of myeloperoxidase (MPO). These results indicate that neither macrophages nor neutrophils are chemoattracted to intact cysts, yet both are capable of killing the cysts. Macrophages killed the cysts by phagocytosis, whereas neutrophils killed cysts through the secretion of MPO.     

Viability of Echinococcus granulosus cysts in mice following cultivation in vitro.

The viability of hydatid cysts developed in vitro for 90 days was assessed by implantation into mice. Cysts removed from mice at 270 days post-infection (p.i.) increased their size 13.5-fold and contained several brood capsules containing protoscoleces. Thus, cysts remain viable after prolonged in vitro culture. The implantation in mice of 15 cysts developed in vitro yielded an average of 10 cysts per mouse, which is indicative of a high survival rate in these experimental infections. The ultrastructural study of cysts recovered from mice 270 days p.i. showed that the germinal membrane was more compact than before implantation and several layers of tegumental cells had developed. Observations of cysts removed from mice indicated that the plasma membrane surrounding microtriches had prolongations opening into the laminated layer.     

The effect of in vitro growth conditions on the resistance of Acanthamoeba cysts

Despite increasing concerns of direct pathogenicity and/or their role as hosts for other microorganisms there are currently no standard methods for the inactivation of amoebae that belong to the genus Acanthamoeba. Methods used to grow amoebae and produce cysts for these tests may be important as they can dramatically modify cyst susceptibility. We compared resistance of cysts produced from trophozoites grown in peptone-yeast extract-glucose broth or by feeding on HEp-2 cells and then encysted in Neff's medium. We observed that trophozoites grown using HEp-2 cells as a nutrient source produce cysts that are significantly more resistant to SDS and to most biocides tested, including heat. Increased resistance is likely due to a higher proportion of mature cysts presenting thicker cell walls as demonstrated using transmission electron microscopy. This was confirmed by calcofluor white staining demonstrating higher cellulose content in cysts produced from trophozoites grown using HEp-2 cells as a feeding source. These results demonstrate that not only methods used to produce cysts from trophozoites are critical, but that methods used to grow trophozoites before encystment should also be chosen carefully. This should be taken into account for the development of protocols to evaluate biocides and antimicrobials against amoebal cysts.     

Survival of Legionella pneumophila within cysts of Acanthamoeba polyphaga following chlorine exposure

The association between Legionella pneumophila and the free-living amoeba Acanthamoeba polyphaga was studied. Intracellular growth of L. pneumophila within amoebic trophozoite was confirmed by kinetic growth experiments, light and electron microscopy. Cysts produced from infected trophozoites were found to protect the legionellas from at least 50 mg/l free chlorine. The ability of L. pneumophila to survive within the cysts of A. polyphaga is suggested as a possible mechanism by which the organism evades disinfection and spreads to colonize new environments.     

The lethal effects of biguanides on cysts and trophozoites of Acanthamoeba castellanii

The effects of a range of biocides on trophozoite and encysted forms of Acanthamoeba castellanii were investigated. Viable acanthamoebae were enumerated by a plaque assay technique. The cyst form of Acanthamoeba castellanii was more resistant to all biocides tested than the trophozoite form. Of the biocides tested, chlorhexidine diacetate (CHA) and polyhexamethylene biguanide (PHMB) were the most effective. Their lethal effects were time- and concentration-dependent. CHA was very effective when formulated in 0.1% EDTA combined with Tris buffer pH 7.8 whereas PHMB activity was reduced by 0.1% EDTA. Three per cent dimethylsulphoxide (DMSO) enhanced the activity of CHA but not of PHMB.     

Survival of Legionella pneumophila within cysts of Acanthamoeba polyphaga following chlorine exposure

The association between Legionella pneumophila and the free-living amoeba Acanthamoeba polyphaga was studied. Intracellular growth of L. pneumophila within amoebic trophozoite was confirmed by kinetic growth experiments, light and electron microscopy. Cysts produced from infected trophozoites were found to protect the legionellas from at least 50 mg/l free chlorine. The ability of L. pneumophila to survive within the cysts of A. polyphaga is suggested as a possible mechanism by which the organism evades disinfection and spreads to colonize new environments.     

Enhanced killing of Acanthamoeba cysts with a plant peroxidase-hydrogen peroxide-halide antimicrobial system

The activity of H(2)O(2) against the resistant cyst stage of the pathogenic free-living amoeba Acanthamoeba was enhanced by the addition of KI and either horseradish peroxidase or soybean peroxidase or, to a lesser degree, lactoperoxidase. This resulted in an increase in the cysticidal activity of 3% (wt/vol) H(2)O(2), and there was >3-log killing in 2 h, compared with the 6 h required for comparable results with the peroxide solution alone (P < 0.05). With 2% H(2)O(2), enhancement was observed at all time points (P < 0.05), and total killing of the cyst inoculum occurred at 4 h, compared with 6 h for the peroxide alone. The activity of sublethal 1% H(2)O(2) was enhanced to give 3-log killing after 8 h of exposure (P < 0.05). No enhancement was obtained when KCl or catalase was used as a substitute in the reaction mixtures. The H(2)O(2) was not neutralized in the enhanced system during the experiments. However, in the presence of a platinum disk used to neutralize H(2)O(2) in contact lens care systems, the enhanced 2% H(2)O(2) system gave 2.8-log killing after 6 h or total cyst killing by 8 h, and total neutralization of the H(2)O(2) occurred by 4 h. In contrast, 2% H(2)O(2) alone resulted in <0.8-log killing of cysts in the presence of the platinum disk due to rapid (<1 h) neutralization of the peroxide. Our observations could result in significant improvement in the efficacy of H(2)O(2) contact lens disinfection systems against Acanthamoeba cysts and prevention of acanthamoeba keratitis.     

Viability of dinoflagellate cysts after the passage through the copepod gut

Several dinoflagellate species form nonmotile, thick-walled resting cysts in their life cycle. Cysts can be ingested by planktonic and benthic organisms, but there is scarce information concerning their survival after the passage through the digestive apparatus of the grazers. We tested the germination capability of cysts produced by two neritic dinoflagellates, Scrippsiella trochoidea (F. Stein) A.R. Loeblich and Scrippsiella ramonii Montresor, after their ingestion by four copepod species. Experiments have been carried out with four species: Acartia clausi Giesbrecht, 1889; Centropages typicus Kröyer, 1849; Temora stylifera Dana, 1849; and Clausocalanus lividus Frost and Fleminger, 1968. Copepods were fed either with motile cells or cysts, and feeding and clearance rates were estimated for A. clausi, C. lividus and T. stylifera. Grazing rates on both dinoflagellates was much higher for vegetative cells than for cysts. Resting cysts were isolated from the faecal pellets and incubated to test their germination capability. S. trochoidea cysts eaten by C. typicus and T. stylifera showed a high germination rate, while cysts of the same species were not viable after the passage through the gut of A. clausi and C. lividus. In contrast, S. ramonii cysts were never able to germinate after being ingested by copepods. The observed variation in viability among the two cyst types and the different survival rates observed for S. trochoidea cysts might be related to differences in cyst morphology and to differences in the digestive process among the tested copepod species.     

Viability of Giardia cysts suspended in lake, river, and tap water

Numerous waterborne outbreaks of giardiasis have occurred since 1965, yet little or no information has been reported on the viability of Giardia cysts in different aquatic environments. We have studied the viability of Giardia muris cysts suspended in lake, river, and tap water, while also monitoring water temperature, dissolved oxygen, pH, and other water quality parameters. Fecal pellets containing G. muris cysts were placed in glass vials covered with filter paper and exposed to (i) lake water at 15 ft (ca. 4.6 m) and 30 ft (ca. 9.2 m), (ii) river water, (iii) tap water, and (iv) distilled water stored under laboratory conditions. At 3, 7, 14, 28, 56, and 84 days, two vials from each environment were removed, and cyst viability was determined by (i) fluorogenic dye exclusion, (ii) production of giardiasis in an animal, and (iii) cyst morphology by Nomarski microscopy. In the fall, the cysts suspended at 30 ft in lake water remained viable for up to 56 days whereas cysts stored at 15 ft were nonviable after day 28. The G. muris cysts exposed to river water remained viable up to 28 days as determined by the production of giardiasis in mice. G. muris cysts suspended in tap water showed no signs of viability after 14 days, while cysts serving as controls (exposed to refrigerated distilled water) remained viable for up to 56 days. In the winter, Giardia cysts suspended in either lake or river water were viable for 56 to 84 days whereas cysts exposed to tap water were nonviable by day 14.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of a real-time PCR assay for quantification of Acanthamoeba trophozoites and cysts

Free-living amoebae have been found to be a reservoir for various pathogenic bacteria in aquatic environments. For example, the Acanthamoeba genus renders possible the intracellular multiplication of Legionella pneumophila, which is responsible for legionellosis. It consequently matters to quantify Acanthamoeba cells and thereby enhance our assessment of the risk of contamination. The classical microbiological method of quantification relies on amoebae growth and most probable number calculation. We have developed a real-time PCR assay based on a TaqMan probe that hybridizes onto 18S rDNA. This probe is specific to the Acanthamoeba genus. The assay was successful with both the trophozoite and the cyst forms of Acanthamoeba. Highly sensitive, it proved to permit detection of fewer than 10 cells, even those that are not easily cultivable, such as the cyst forms.     

Resistance of Acanthamoeba castellanii cysts to physical,chemical, and radiological conditions

Resistance of Acanthamoeba castellanii cysts to disinfection agents, antimicrobial agents, heat, freeze-thawing, ultraviolet radiation (UV), gamma irradiation, and cellulase were evaluated in vitro. Following exposure to different agents, the cysts were removed and cultured for A. castellanii trophozoites for 3-14 days. Solutions containing 20% isopropyl alcohol or 10% formalin effectively killed A. castellanii cysts. Hydrogen peroxide (3%, AOSept Disinfectant) effectively killed A. castellanii cysts after 4 hr of exposure. Polyhexamethylene biguanide (0.02%), clotrimazole (0.1%), or propamidine isethionate (Brolene) were effective in killing A. castellanii cysts in vitro. Acanthamoeba castellanii cysts were resistant to both 250 K rads of gamma irradiation and 800 mJ/cm2 of UV irradiation. Excystment of trophozoites was accelerated after exposure to 10, 100, and, 1,000 units of cellulase. These results suggest that A. castellanii cysts benefit by enhanced survival because of their resistance to very harsh environmental conditions.     

Viability of Giardia cysts suspended in lake, river, and tap water.

Numerous waterborne outbreaks of giardiasis have occurred since 1965, yet little or no information has been reported on the viability of Giardia cysts in different aquatic environments. We have studied the viability of Giardia muris cysts suspended in lake, river, and tap water, while also monitoring water temperature, dissolved oxygen, pH, and other water quality parameters. Fecal pellets containing G. muris cysts were placed in glass vials covered with filter paper and exposed to (i) lake water at 15 ft (ca. 4.6 m) and 30 ft (ca. 9.2 m), (ii) river water, (iii) tap water, and (iv) distilled water stored under laboratory conditions. At 3, 7, 14, 28, 56, and 84 days, two vials from each environment were removed, and cyst viability was determined by (i) fluorogenic dye exclusion, (ii) production of giardiasis in an animal, and (iii) cyst morphology by Nomarski microscopy. In the fall, the cysts suspended at 30 ft in lake water remained viable for up to 56 days whereas cysts stored at 15 ft were nonviable after day 28. The G. muris cysts exposed to river water remained viable up to 28 days as determined by the production of giardiasis in mice. G. muris cysts suspended in tap water showed no signs of viability after 14 days, while cysts serving as controls (exposed to refrigerated distilled water) remained viable for up to 56 days. In the winter, Giardia cysts suspended in either lake or river water were viable for 56 to 84 days whereas cysts exposed to tap water were nonviable by day 14.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rheology of sodium hyaluronate saline solutions for ophthalmic use

The aim of this work was to investigate the rheological properties of different saline solutions of sodium hyaluronate (NaHA) with special interest for medical applications. The experimental results were compared with literature data for commercial ophthalmic viscosurgical devices (OVDs) used in cataract surgery. We offer some tools to tailor the rheological behavior of OVDs for different purposes. We have investigated to which extent surgical requirements can be fulfilled by adjusting either the molecular weight of NaHA or its concentration, parameters that are in some respects equivalent but not in others. Furthermore, we demonstrate that moduli and complex viscosities of NaHA saline solutions are adequately falling on master curves, using either empirical or calculated shift factors, the latest ones being based on a modified Rouse model.     

Aspects of the mechanisms of action of biguanides on trophozoites and cysts of Acanthamoeba castellanii

A non-radioactive method was used to investigate the uptake by Acanthamoeba castellanii of chlorhexidine diacetate (CHA) and polyhexamethylene biguanide (PHMB). Based on the Giles et al. (1974) hypothesis, the uptake of CHA by trophozoites appeared to be of the L3 pattern whereas that of cysts was C2. Unlike CHA, trophozoites took up PHMB with an L2 pattern at low concentrations followed by a C-type pattern at higher concentrations, the uptake by cysts was found to be of the C2 pattern with a plateau effect at high concentrations. A diphasic leakage effect was found in trophozoites whereas a relatively low peak of maximal leakage occurred from cysts treated with high biocide concentrations. The amount of pentose release depended on the formulation ingredients. No correlation between pentose leakage and trophozoicidal or cysticidal activity was found.     

Interaction of Escherichia coli K1 and K5 with Acanthamoeba castellanii trophozoites and cysts

The existence of symbiotic relationships between Acanthamoeba and a variety of bacteria is well-documented. However, the ability of Acanthamoeba interacting with host bacterial pathogens has gained particular attention. Here, to understand the interactions of Escherichia coli K1 and E. coli K5 strains with Acanthamoeba castellanii trophozoites and cysts, association assay, invasion assay, survival assay, and the measurement of bacterial numbers from cysts were performed, and nonpathogenic E. coli K12 was also applied. The association ratio of E. coli K1 with A. castellanii was 4.3 cfu per amoeba for 1 hr but E. coli K5 with A. castellanii was 1 cfu per amoeba for 1 hr. By invasion and survival assays, E. coli K5 was recovered less than E. coli K1 but still alive inside A. castellanii. E. coli K1 and K5 survived and multiplied intracellularly in A. castellanii. The survival assay was performed under a favourable condition for 22 hr and 43 hr with the encystment of A. castellanii. Under the favourable condition for the transformation of trophozoites into cysts, E. coli K5 multiplied significantly. Moreover, the pathogenic potential of E. coli K1 from A. castellanii cysts exhibited no changes as compared with E. coli K1 from A. castellanii trophozoites. E. coli K5 was multiplied in A. castellanii trophozoites and survived in A. castellanii cysts. Therefore, this study suggests that E. coli K5 can use A. castellanii as a reservoir host or a vector for the bacterial transmission.