Free-living amoebae: characterisation, inactivation and pathogenicity, with special reference to human ocular infection in South Africa

Niszl, Ingrid Anne
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In this thesis, collection and other details of strains of Acanthamoeba and Mastigina species are recorded for archival purposes for southern African research reference. This information is mainly derived from 14 of 40 southern African patients suspected of having acanthamoebic keratitis. The high prevalence in the environment of Gauteng of Acanthamoeba, including highly virulent strains, is demonstrated. The pathogenicity of clinical and environmental Acanthamoeba and Mastigina isolates was compared in vitro, using two different mammalian cell lines. The time taken by the amoebae to destroy cell cultures completely was shown to be largely dependent on the size of the amoebic inoculum. The use of mammalian cell cultures was shown to be an accurate, rapid and repeatable means of assaying the cytopathic effect of strains of Acanthamoeba. The effects of contact lens disinfecting solutions on strains of Acanthamoeba and Mastigina were assessed. Not all solutions used in the study were effective: some intended for hard and gas-permeable contact lenses proved more satisfactory than those for soft contact lenses. Manufacturers should be aware of the killing time for Acanthamoeba and Mastigina species by contact lens solutions, and should provide appropriate guidelines for their use. The sensitivity to drugs of isolates of Acanthamoeba and Mastigina was compared. Mastigina species was sensitive to amphotericin B, mefloquine-hydrochloride and polymyxin B sulphate, whereas polyhexamethylene biguanide or chlorhexidine should be the drug of choice for keratitis when Acanthamoeba is isolated. Light and electron microscopy of cysts was used to characterise strains of Acanthamoeba and Mastigina. The author obtained consistently satisfactory results using a simple technique that was adapted to facilitate embedding amoebae for transmission electron microscopy. Morphological similarities were apparent between cysts of several isolates. Cellulose acetate and polyacrylamide gel electrophoresis were conducted to characterise strains of Acanthamoeba and a strain of Mastigina. In some instances, diversity in isoenzyme patterns for some enzymes was found in certain strains assigned to the same species, while in other cases similarities were apparent. Restriction fragment length polymorphisms, using nine enzymes, differentiated seven strains of Acanthamoeba into five groups. These results confirm those obtained electrophoretically.