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Purpose: To determine whether increased rates of Acanthamoeba keratitis (AK) are due to changes in municipal water treatment or to emergence of a more pathogenic strain of Acanthamoeba., Methods: Previous sequence analysis of the 18S ribosomal DNA of Acanthamoeba isolates resulted in the identification of 15 different genotypic classes. These analyses indicate that AK cases are associated predominantly ( approximately 97%) with a single genotype (designated T4) of Acanthamoeba and rarely with other genotypes (eg, T3 and T11). In this study, we test the hypothesis that a new or more pathogenic genotype of Acanthamoeba is the cause of the recent surge in AK., Results: We determined the genotype of 15 Acanthamoeba sp. isolates from AK cases associated with this outbreak using sequence analysis of a region of the 18S ribosomal DNA. Our results indicate that these isolates are predominantly genotype T4 (87%), with the remaining isolates being genotype T3 (13%). Both genotypes have previously been observed in AK cases., Conclusions: There is no support for the hypothesis that the current AK outbreak is associated with infection by a new more pathogenic Acanthamoeba genotype. In addition, these results offer support for the hypothesis that the increased AK incidence may be because of changes in water treatment protocols leading to increased bacterial colonization of the water supply and subsequent increases of already present Acanthamoeba sp, ultimately culminating in an increase of AK cases.