Molecular diagnosis of Acanthamoeba keratitis: evaluation in rat model and application in suspected human cases
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Acanthamoeba keratitis (AK) is a progressive corneal infection that demands rapid and sensitive techniques for diagnosis to avoid risk of visual impairment. We evaluated two DNA extraction techniques and a semi-nested-PCR (snPCR) targeting the 18S rRNA gene to detect Acanthamoeba cysts and trophozoites. The most effective protocol was evaluated in samples of corneal scrapings and biopsies from an AK rat model and applied to diagnosis of human cases of AK. DNA extraction performed with a commercial kit based on DNA binding to magnetic beads was more efficient than a method based on alkaline lysis, allowing the detection of one trophozoite and one cyst of Acanthamoeba in samples prepared from cultures. This technique and sn-PCR were applied in corneal scrapings of rats experimentally infected with Acanthamoeba (n = 6), resulting in 100% of positivity, against 16.7% (n = 6) of positive identification in culture method using non-nutrient agar (NNA) with Escherichia coli. Corneal biopsies from rats were also tested (n = 6) and resulted in positivity in all samples in both molecular and culture methods. Eight out of ten presumptive human cases of Acanthamoeba keratitis were also confirmed by sn-PCR of scrapping samples, while the culture method was positive in only four cases. We discuss that animal model of AK can be an efficient tool to validate diagnostic methods and conclude that DNA extraction with the kit and snPCR protocol described here is an effective alternative for diagnosis of AK.
KeywordsAcanthamoeba keratitis Rat model Diagnosis Semi-nested PCR Culture
The authors thank CNPq, Fundação Araucária, and PRPq/UFMG for financial support. We also thank Ophthalmology Sector staff from Cassiano Antônio de Moraes University Hospital for providing the human corneal scrapings used in this study.
Compliance with ethical standards
All procedures with animal were conducted under accepted guidelines for the care and use of laboratory animals for research and approved by the Ethical Committee for Animal Experimentation of the Biological Sciences Sector of Federal University of Paraná, Brazil, protocol number 420b.
The use of information on the diagnosis of patients suspected of Acanthamoeba keratitis was approved by the Ethical Committee of Health Sciences Center of Federal University of Espírito Santo, Vitória, ES, Brazil (protocol number 006/07), and followed the tenets of Declaration of Helsinki.
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