This study was approved by the local ethics committee (the Federal University of São Paulo [UNIFESP] 0344/12HE) and was conducted in full compliance with the current biosecurity standards.
Acanthamoeba cysts were obtained from corneal tissue surface of two female patients, contact lens wearers, whose primary clinical symptoms were contact lens wear intolerance, pain, photophobia, and tear production. The clinical isolates of
Acanthamoeba spp were designated as isolate 01 and isolate 02. The degree of corneal infection from both clinical cases was categorized as severe, using criteria described previously by Vital et al.
13 Collection of clinical samples was conducted in accordance with the tenets of the Declaration of Helsinki, and prior informed consent was obtained. Primary isolation of
Acanthamoeba cysts was obtained on nonselective agar medium, used in the laboratory diagnostic routine to isolate free living amoeba from corneal samples, as described previously.
14,15 The reference strain
Acanthamoeba castellanii from the American Type Culture Collection (ATCC 30011) was used as experimental control. The average diameter of
Acanthamoeba cysts from isolate 01 and isolate 02 was 17.35 and 16.55 μm, respectively, providing preliminary morphologic information to characterize
Acanthamoeba cysts from isolate 01 and isolate 02 as members of group II, according the classification defined by Pussard and Pons.
16 Molecular characterization of clinical isolates of
Acanthamoeba spp was based on the nearly complete 18S rRNA gene sequence. The reference strain
A. castellanii (genotype T4, ATCC 30011) and Milli-Q ultrapure water (Milli-Q; Millipore GmbH, Eschborn, Germany) were used as positive and negative controls, respectively. Genomic DNA was extracted with an UltraClean Tissue and Cells DNA Isolation Kit (MoBio Laboratories, Carlsbad, CA) and the nucleic acid content was quantified by spectrophotometry at 260 nm using a BioPhotometer (Eppendorf, Hamburg, Germany). The purity of the DNA extracted was standardized in 1.8 considering the ratio of A260/A280. PCRs were performed using five sets of universal eukaryotic primers, as shown in
Table 1. Amplicons were generated in a 50 μL reaction volume containing 1× Q5 Hot Start High-Fidelity Master Mix (New England Biolabs, Beverly, MA), both forward and reverse primers (0.2 μM) and DNA template at final concentration of 1 ng/μl. The standardized amplification profile was performed with an initial denaturation step at 98°C for 2 minutes followed by 30 cycles at 98°C for 30 seconds (denaturation), 60°C for 30 seconds (annealing), 74°C for 1 minute (extension), and a final extension at 74°C for 6 minutes. PCR products to be sequenced were purified with the PureLink PCR Purification Kit (Invitrogen Life Technologies, Carlsbad, CA). Both strands of each amplicon were sequenced and data were processed with the BioEdit Sequence Alignment Editor version 7.2.0 (Ibis Biosciences, Carlsbad, CA).
21 The nucleotide sequences obtained were compared to the GenBank data library with BLAST software.
22 The 18S rRNA gene sequences were deposited in the GenBank database under accession numbers KF318460 to KF318462.