title: Molecular diagnosis of Acanthamoeba keratitis: evaluation in rat model and application in suspected human cases.
abstract: 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.
authors.affiliation: Departamento de Análises Clínicas e Toxicológicas, Faculdade de Farmácia, Universidade Federal de Minas Gerais, Av. Presidente Antônio Carlos, 6627, Belo Horizonte, Minas Gerais, 31270-901, Brazil. adrincosta@gmail.com.
authors.forename: Adriana Oliveira
authors.initials: AO
authors.lastname: Costa
authors.affiliation: Departamento de Patologia, Universidade Federal do Espírito Santo, Vitória, Espírito Santo, Brazil.
authors.forename: Cinthia
authors.initials: C
authors.lastname: Furst
authors.affiliation: Departamento de Patologia Básica, Setor de Ciências Biológicas, Universidade Federal do Paraná, Curitiba, Paraná, Brazil.
authors.forename: Lucas Oliveira
authors.initials: LO
authors.lastname: Rocha
authors.affiliation: Departamento de Análises Clínicas e Toxicológicas, Faculdade de Farmácia, Universidade Federal de Minas Gerais, Av. Presidente Antônio Carlos, 6627, Belo Horizonte, Minas Gerais, 31270-901, Brazil.
authors.forename: Cecília
authors.initials: C
authors.lastname: Cirelli
authors.affiliation: Departamento de Análises Clínicas e Toxicológicas, Faculdade de Farmácia, Universidade Federal de Minas Gerais, Av. Presidente Antônio Carlos, 6627, Belo Horizonte, Minas Gerais, 31270-901, Brazil.
authors.forename: Carolina Neris
authors.initials: CN
authors.lastname: Cardoso
authors.affiliation: Departamento de Patologia Básica, Setor de Ciências Biológicas, Universidade Federal do Paraná, Curitiba, Paraná, Brazil.
authors.forename: Fagner Salmazo
authors.initials: FS
authors.lastname: Neiva
authors.affiliation: Departamento de Patologia, Universidade Federal do Espírito Santo, Vitória, Espírito Santo, Brazil.
authors.forename: Cynara Oliveira
authors.initials: CO
authors.lastname: Possamai
authors.affiliation: Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
authors.forename: Daniel
authors.initials: D
authors.lastname: de Assis Santos
authors.affiliation: Departamento de Patologia Básica, Setor de Ciências Biológicas, Universidade Federal do Paraná, Curitiba, Paraná, Brazil.
authors.forename: Vanete
authors.initials: V
authors.lastname: Thomaz-Soccol
mesh_terms: D000048:Acanthamoeba / Q000235:genetics / Q000302:isolation & purification*; D015823:Acanthamoeba Keratitis / Q000175:diagnosis* / Q000469:parasitology; D000818:Animals; D003315:Cornea / Q000469:parasitology; D016054:DNA, Protozoan / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D023421:Models, Animal; D016133:Polymerase Chain Reaction / Q000379:methods; D051381:Rats; D012680:Sensitivity and Specificity; D053058:Trophozoites
chemical_list: D016054:DNA, Protozoan
keywords: Acanthamoeba keratitis; Culture; Diagnosis; Rat model; Semi-nested PCR