Isolation and molecular characterization of Acanthamoeba from patients with keratitis in Spain

In order to improve our knowledge on the epidemiology of amoebic keratitis, as well as the identification of Acanthamoeba isolates, we have isolated Acanthamoeba spp. from five symptomatic patients in Spain in the present study. All isolates were grown in axenic liquid medium, with only one exception. The morphology of these isolates were characterized by optical and scanning electron microscopy. Their structural features corresponded to those of amphizoic amoebae (namely Acanthamoeba spp.). The molecular characterization of the five Acanthamoeba isolates yielded six sequences. Almost complete 18S rRNA gene sequences (>2000 bp) were obtained from three isolates and partial sequences (∼1500 bp) from the other two. A robust phylogenetic analysis based on the almost complete 18S rRNA sequence showed that four isolates belonged to the T4 genotype and the other one to the T3 genotype. However, all isolates were identified as T4 genotype using the ASA.S1 fragment. As previously suggested by other researchers, only a robust phylogenetic approach may be helpful in identifying Acanthamoeba genotypes. In addition, new data on the phylogenetic relationships among the Acanthamoeba genotypes is provided and discussed.