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2010 OMIG, Abstract 7

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Discriminative profile between Acanthamoeba T4 and non-T4 genotypes by 18S rDNA gene fingerprint
F.R.S. Carvalho, A.S.  Foronda, D. de Freitas, Federal University of Sao Paulo, Dept. of Ophthalmology

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 (n = 19), 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. Restriction enzyme analysis was performed and fingerprinting profile of the amplimer ASA.S1was determined by electrophoresis.
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.

Disclosure Code: FAPESP (Grant 08/53969-0), CAPES (PNPD Scientific Program), CNPq.

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