Identification and drug sensitivities of Acanthamoeba species causing keratitis : a thesis presented in partial fulfillment of the requirements for the degree of Master of Science in Microbiology at Massey University, Palmerston North, New Zealand

Abstract

Acanthamoebic keratitis is a distinct, vision-threatening ophthalmological condition, the incidence of which is increasing with increased usage of contact lenses. Diagnosis can be difficult and chemotherapeutic treatment is prolonged and often ineffective. It is therefore desirable to discover a quick and accurate means of diagnosing acanthamoebic keratitis, and to gain knowledge regarding which chemotherapeutic agents are most effective against acanthamoebic keratitis. The first goal of this thesis was to examine ten DNA extraction procedures and determine their effectiveness in extracting DNA from Acanthamoeba cells. Of these ten methods, four (2, 7, 8, 10) could be performed in less than one day and showed consistent results in PCR reactions. The second goal of this thesis involved the application of arbitrarily primed polymerase chain reaction (AP-PCR) in an attempt to type and group strains of Acanthamoeba species. Examination of 16 isolates with primers AP0l, AP02, AM1, AM2, P1 and P2, showed each of the banding patterns, resulting from AP-PCR analysis, were unique to the isolate tested. Further, there were few bands which occurred in more than one isolate, with insufficient similarities to form groupings of isolates. Two chemotherapeutic agents were selected for a preliminary study into drug sensitivities in Acanthamoeba species and strains. The first of these was Baquacil (20% polyhexamethylene biguanide (PHMB)), and the second was Brolene (0.1% propamidine isethionate). Within 48 hours 97% of all isolates tested reached zero viability at a concentration of 0.05% PHMB, and 100% of isolates tested reached zero viability at 0.1% PHMB. The results of this study would suggest that the concentration of PHMB be at least 0.05% when used to treat acanthamoebic keratitis. Within 48 hours of exposure to 0.1% propamidine isethionate (Brolene), only 30% of all isolates tested reached zero viability. However, 60% of isolates tested showed at least 80% reduction in viability within 48 hours of exposure to 0.1% propamidine isethionate. The ultimate goal of this thesis was to form groups of isolates using PCR and drug sensitivities and to discover any correlation between these groups. The results of AP- PCR analysis however suggests a high genetic heterogeneity within the Acanthamoeba genus, thus preventing any correlation with drug sensitivity tests.