June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Phylogenetic study of Acanthamoeba genotypes determined by 18S rDNA gene analysis
Author Affiliations & Notes
  • Denise Freitas
    Ophthalmology, Federal University of Sao Paulo, Sao Paulo, Brazil
  • Felipe Marques de Carvalho Taguchi
    Ophthalmology, Federal University of Sao Paulo, Sao Paulo, Brazil
  • Linda Carrijo-Carvalho
    Ophthalmology, Federal University of Sao Paulo, Sao Paulo, Brazil
  • Viviane Peracini
    Ophthalmology, Federal University of Sao Paulo, Sao Paulo, Brazil
  • Annette Foronda
    Ophthalmology, Federal University of Sao Paulo, Sao Paulo, Brazil
  • Fábio Ramos
    Ophthalmology, Federal University of Sao Paulo, Sao Paulo, Brazil
  • Footnotes
    Commercial Relationships Denise Freitas, None; Felipe Marques de Carvalho Taguchi, None; Linda Carrijo-Carvalho, None; Viviane Peracini, None; Annette Foronda, None; Fábio Ramos, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 2922. doi:
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      Denise Freitas, Felipe Marques de Carvalho Taguchi, Linda Carrijo-Carvalho, Viviane Peracini, Annette Foronda, Fábio Ramos, ; Phylogenetic study of Acanthamoeba genotypes determined by 18S rDNA gene analysis. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2922.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Acanthamoeba keratitis (AK) is a severe condition with sight-threatening potential. Although the genus Acanthamoeba has been classified into 16 different genotypes based on rDNA sequence analyses (T1-T16), T4 genotype has been most related with ocular infection. In order to provide an accurate discriminative tool to differentiate Acanthamoeba T4 and non-T4 genotypes, the 18S ribosomal RNA gene (18S rDNA) was studied and a fingerprinting profile was developed

Methods: The research was conducted in accordance with the tenets of the Declaration of Helsinki. Approval of the study was obtained from the local institutional review boards. Clinical isolates of Acanthamoeba were obtained from corneal scraping of different patients, while A. castellanii strain (T4 genotype) was obtained from American Type Culture Collection (ATCC 30011). All amoebae were grown without shaking, at room temperature, in 5 ml of Neff medium for 72 hours. The nuclear DNA was extracted and a specific-fragment within 18S rDNA gene of Acanthamoeba spp, denominated ASA.S1, was amplified by PCR reaction.

Results: A PCR-amplified 18S rDNA Acanthamoeba-specific product followed by restriction analysis were able to provide a discriminative profile between Acanthamoeba T4 and non-T4 genotypes. In addition, two Acanthamoeba isolates investigated were classified as non-T4 genotype

Conclusions: Since the earlier detection of Acanthamoeba species/ genotypes followed by a specific therapeutic procedure could avoid the spread of infection to deep stroma and decrease the severity of infection, the present study showed a reproductive method based on molecular biology tool to differentiate Acanthamoeba T4 genotype, which is most related with AK cases, and non-T4 genotypes. Thus, the application of 18S rDNA gene fingerprint procedure could be used as useful molecular marker in the differentiation of Acanthamoeba genotypes and open perspectives to an earlier detection of pathogenic Acanthamoeba strains in corneal infections. Financial support: FAPESP (Grant 08/53969-0), CAPES (PNPD Scientific Program)

Keywords: 402 Acanthamoeba • 537 gene screening • 573 keratitis  
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