Encystation of Acanthamoeba castellanii: Dye uptake for assessment by flow cytometry and confocal laser scanning microscopy

Connell, Christopher; Rutter, Andrew J.; Hill, Bethan; Suller, M. T. E.; Lloyd, David

doi:10.1046/j.1365-2672.2001.01296.x

Cited by 17 publications

(13 citation statements)

References 15 publications

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“…Growth phase trophozoites were harvested from the culture flasks and washed 2 times with phosphate buffered saline (PBS) and resuspended in 10 mL of encystment medium containing 50 mm of MgCl 2 at a final concentration of 1 × 10 6 trophozoites/mL 12. The trophozoites were incubated at 4°C for 1 week for encystment.…”

Section: Methodsmentioning

confidence: 99%

Amoebicidal Activities of Alexidine Against 3 Pathogenic Strains of Acanthamoeba

Alizadeh

Neelam

Cavanagh

2009

Eye &Amp; Contact Lens: Science &Amp; Clinical Practice

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Objectives Effective pharmacotherapy for Acanthamoeba keratitis has been hampered because of the marked resistance of various stains to a variety of antimicrobial agents. In view of the fact that topical Brolene (propamidine isethionate) and neosporin are currently considered to be the first-line medical treatment of choice in Europe, we sought to determine whether Alexidine is equally effective, because the latter drug is more readily available in the United States. Methods Trophozoites and cysts from 3 pathogenic corneal isolates (A. castellanii, A. polyphaga, and A. rhysodes) were incubated in peptone-yeast extract-glucose medium containing different concentrations of Alexidine for 24 hr. The number of trophozoites was counted by hemocytometer. The cysts were plated in to nonnutrient agar plates precoated with Escherichia coli and observed for viability or excystment over a period of 2 weeks. The capacity of different concentrations of Alexidine to induce cytolysis of corneal epithelial cells was tested in vitro. Chinese hamster corneas were treated with 5 μL of Alexidine topically, every hour; 6 times a day and the corneas were stained with fluorescein to asses the epithelial defects in vivo. Results Alexidine was effective in killing the trophozoites at a concentration of 10 μg/mL. However, a higher concentration of Alexidine (100 μg/mL) is required to kill Acanthamoeba cysts and the cytotoxic activities of Alexidine are comparable with chlorhexidine. We have also demonstrated that both Alexidine and chlorhexidine at 100 μg/mL induced significant cytopathic effect on the corneal epithelial cells in vitro. In vivo results indicate that Alexidine at a concentration of 100 μg/mL is less toxic than chlorhexidine when applied topically to the Chinese hamster cornea. Conclusions Our study has identified Alexidine as a novel anti-Acanthamoeba drug and suggests that Alexidine may be an effective therapeutic option because of its potency and low toxicity to the corneal tissues when applied topically in vivo.

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Section: Methodsmentioning

confidence: 99%

Amoebicidal Activities of Alexidine Against 3 Pathogenic Strains of Acanthamoeba

Alizadeh

Neelam

Cavanagh

2009

Eye &Amp; Contact Lens: Science &Amp; Clinical Practice

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“…Mechanisms of biocide action have been extensively studied recently [49][50][51][52][53][54]. It is thought that resistance that begins to increase after 8 h is caused by the development of an acidinsoluble ectocyst wall that contains protein and acts as a nonpermeable barrier [55].…”

Section: Biocidal Resistance and Treatmentmentioning

confidence: 99%

Recent Advances in the Treatment ofAcanthamoebaKeratitis

Kumar

Lloyd

2002

CLIN INFECT DIS

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Infection of the eye caused by Acanthamoeba species constitutes a burgeoning and unsolved problem. Of individuals with Acanthamoeba keratitis, 85% wear contact lenses; abrasion of the cornea is implicated. Corneal infection often can be prevented by good lens care and hygiene. Severe Acanthamoeba keratitis often can be very difficult to treat; surgery can be less than successful and may lead to further problems. The encysted stage in the life cycle of Acanthamoeba species appears to cause the most problems; many biocides are ineffective in killing the highly resistant cysts. Combination therapy--that is, use of 2 or 3 biocides, sometimes with antibacterial antibiotics--appears to work best. Recurrence is common if treatment is stopped prematurely. Immunologic methods are being investigated as a form of prevention, and oral immunization of animals recently has been successful in the prevention of Acanthamoeba keratitis by inducing immunity before infection occurs. Immunization thus may eventually become the best approach for reduction of the incidence of amebic infection in humans.

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“…This would provide a rapid method for assessing encystation progress. Flow cytometry has been used to track DNA content during encystation in E. invadens [16] and Giardia intestinalis [17] and chitin accumulation during encystation in Acanthamoeba castellani [18]. To the best of our knowledge, this is the first report describing a flow cytometry-based method to assess Entamoeba encystation by simultaneously tracking changes in cell size/shape and chitin.…”

mentioning

confidence: 99%

“…Cells were collected over time, stained with the fluorescent chitin stain, Congo Red [18], fixed, and analyzed by flow cytometry by collecting ten-thousand individual events. In addition to fluorescence, flow cytometers measure the light scattered by single particles at right angles to the laser beam (side scatter, SSC) and in the forward direction (forward scatter, FSC).…”

mentioning

confidence: 99%

Flow cytometric characterization of encystation in Entamoeba invadens

Welter

Sehorn

Temesvari

2017

Molecular and Biochemical Parasitology

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Entamoeba histolytica causes dysentery and liver abscess mostly in countries that lack proper sanitation. Infection is acquired by ingestion of the cyst form in contaminated food or water. E. histolytica does not encyst in vitro; thus, E. invadens, a reptilian parasite that encysts in vitro, has been used as a surrogate. Cysts are small and possess chitin-rich walls. These are characteristics that may be exploited by flow cytometry. We stained encysting E. invadens cells with a fluorescent chitin stain, and analyzed fluorescence and forward scatter by flow cytometry. We demonstrate that flow cytometry can be used to track differentiation, reveal unique cell populations, and evaluate encystation inhibitors.

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Encystation of Acanthamoeba castellanii: Dye uptake for assessment by flow cytometry and confocal laser scanning microscopy

Cited by 17 publications

References 15 publications

Amoebicidal Activities of Alexidine Against 3 Pathogenic Strains of Acanthamoeba

Amoebicidal Activities of Alexidine Against 3 Pathogenic Strains of Acanthamoeba

Recent Advances in the Treatment ofAcanthamoebaKeratitis

Flow cytometric characterization of encystation in Entamoeba invadens

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