March 2012
Volume 53, Issue 14
Free
ARVO Annual Meeting Abstract  |   March 2012
Effects Of Multi-purpose Solutions On The Viability And Encystment Of Clinical Isolates Of Acanthamoeba Determined By Flow Cytometry
Author Affiliations & Notes
  • Masaki Imayasu
    R & D Center, Menicon Co Ltd, Kasugai, Japan
  • Kissaou T. Tchedre
    R & D Center, Menicon Co Ltd, Kasugai, Japan
  • H D. Cavanagh
    Ophthalmology, Univ Texas Southwestern Med Ctr, Dallas, Texas
  • Footnotes
    Commercial Relationships  Masaki Imayasu, Menicon Co., Ltd. (E); Kissaou T. Tchedre, Menicon Co., Ltd. (E); H. D. Cavanagh, Menicon Co., Ltd. (C)
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 6077. doi:
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      Masaki Imayasu, Kissaou T. Tchedre, H D. Cavanagh; Effects Of Multi-purpose Solutions On The Viability And Encystment Of Clinical Isolates Of Acanthamoeba Determined By Flow Cytometry. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6077.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : The encystment process of Acanthamoeba spp is characterized by trophozoite rounding. It was reported that the incidence of Acanthamoeba keratitis (AK) in the United States was related to one specific multi-purpose solution (MPS), COMPLETE Moisture PLUS (CMP), that induced the transformation of Acanthamoeba trophozoites into resistant cyst. In this study, we simultaneously evaluated the effects of MPS on the viability and encystment of clinically isolated Acanthamoeba using flow cytometry.

Methods: : Viability and encystment rate were evaluated using three clinical strains of Acanthamoeba spp isolated from AK patients and Acanthamoeba castellanii (ATCC50514) when treated with four PHMB-based MPSs including CMP. Trophozoites (1.0x105) were exposed to each MPS for 24 hours. After dispensing the cell suspension into two aliquots, one aliquot was stained with 0.004% Congo Red (CR), a fluorescence dye to stain the inner cell wall of cyst, and the other aliquot was stained with a mixture of Congo Red and 1% Sarkosyl (CRS), a detergent to lyse the trophozoites and pseudo-cyst. Flow cytometric analysis of the treated aliquots was carried out on EPICS ALTRA flow cytometer. The encystment rate and disinfecting efficacies (percentage of rounded trophozoites, pseudo-cyst) were calculated by the rates of CR-stained part, CR-non-stained part and CRS-stained part. Ultra-structures of resistant (mature or immature) cyst and pseudo-cyst were observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM).

Results: : Resistant cysts and rounded trophozoites (pseudo-cysts) were stained with CR, whereas native (unrounded) trophozoites were not. Resistant cysts were also stained with CRS unlike pseudo-cysts. No differences were found between the viabilities of clinical isolates and that of ATCC strain; however, three clinical isolates showed higher encystment rates (77.1%, 61.6%, 48.0%) than ATCC strain (43.0%) when treated with CMP. Disinfecting efficacy of each MPS was not directly related to each encystment rate. SEM and TEM observations showed basic differences in the fine structure of pseudo-cysts produced by MPSs and resistant cysts.

Conclusions: : It was suggested that disinfection and encystment of Acanthamoeba are independent phenomenon. Not only high encystment rate but also low disinfecting efficacies of MPS are thought to be associated with the incidence of AK.

Keywords: Acanthamoeba • flow cytometry • microscopy: electron microscopy 
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