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Our institution is the Austrian reference laboratory for <jats:italic>Acanthamoeba<\/jats:italic> diagnostics and the aim of this study was to give an overview of proven cases of <jats:italic>Acanthamoeba<\/jats:italic> infections in Austria during the past 20\u00a0yr. All samples of patients with suspected <jats:styled-content style=\"fixed-case\">AK<\/jats:styled-content> or <jats:styled-content style=\"fixed-case\">GAE<\/jats:styled-content> were screened for <jats:italic>Acanthamoeba<\/jats:italic> spp. by culture and\/or <jats:styled-content style=\"fixed-case\">PCR<\/jats:styled-content> and the detected amoebae were genotyped. Altogether, 154 cases of <jats:styled-content style=\"fixed-case\">AK<\/jats:styled-content> and three cases of <jats:styled-content style=\"fixed-case\">GAE<\/jats:styled-content> were diagnosed. Age of the <jats:styled-content style=\"fixed-case\">AK<\/jats:styled-content> patients ranged from 8 to 82\u00a0yr (mean 37.8) and 58% of the patients were female. Approximately 89% of the <jats:styled-content style=\"fixed-case\">AK<\/jats:styled-content> patients were contact lens wearers, almost all cases were unilateral and 19% of the patients required a keratoplasty. Age of the <jats:styled-content style=\"fixed-case\">GAE<\/jats:styled-content> patients ranged from 2 to 25\u00a0yr (mean 14.7), all were <jats:styled-content style=\"fixed-case\">HIV<\/jats:styled-content>\u2010negative, but two were severely immunosuppressed at the time of diagnosis. The predominant genotype in the <jats:styled-content style=\"fixed-case\">AK<\/jats:styled-content> cases was T4, other genotypes found were T3, T5, T6, T10 and T11. 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