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Detection of Acanthamoeba spp. using carboxylesterase antibody and its usage for diagnosing Acanthamoeba-keratitis.

ABSTRACT: Contact lens usage has contributed to increased incidence rates of Acanthamoeba keratitis (AK), a serious corneal infection that can lead to blindness. Since symptoms associated with AK closely resemble those incurred by bacterial or fungal keratitis, developing a diagnostic method enabling rapid detection with a high degree of Acanthamoeba-specificity would be beneficial. Here, we produced a polyclonal antibody targeting the carboxylesterase (CE) superfamily protein secreted by the pathogenic Acanthamoeba and evaluated its diagnostic potential. Western blot analysis revealed that the CE antibody specifically interacts with the cell lysates and conditioned media of pathogenic Acanthamoeba, which were not observed from the cell lysates and conditioned media of human corneal epithelial (HCE) cells, Fusarium solani, Staphylococcus aureus, and Pseudomonas aeruginosa. High titers of A. castellanii-specific antibody production were confirmed sera of immunized mice via ELISA, and these antibodies were capable of detecting A. castellanii from the cell lysates and their conditioned media. The specificity of the CE antibody was further confirmed on A. castellanii trophozoites and cysts co-cultured with HCE cells, F. solani, S. aureus, and P. aeruginosa using immunocytochemistry. Additionally, the CE antibody produced in this study successfully interacted with 7 different Acanthamoeba species. Our findings demonstrate that the polyclonal CE antibody specifically detects multiple species belong to the genus Acanthamoeba, thus highlighting its potential as AK diagnostic tool.

SUBMITTER: Kim MJ

PROVIDER: S-EPMC8730387 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundAcanthamoeba spp. are the causative agents of a severe keratitis occurring mainly in contact lens wearers. The genus comprises more than 24 species that are currently divided into 20 different genotypes (T1-T20) according to sequence variations in the 18S rRNA gene. The objective of this study was to identify the genotypes and sub-genotypes of Acanthamoeba isolates collected at the Parasitology Laboratory of the Public Health Institute of Chile, the only laboratory in the country where Acanthamoeba screening is performed. This is the first report of genotypic identification of clinical isolates of Acanthamoeba in Chile and one of the few in South America.ResultsIn this study, 114 Acanthamoeba isolates from 76 Acanthamoeba keratitis patients, obtained between 2005-2016, were genotyped. T4 was the predominant genotype; T2 and T11 genotypes, which are scarcely reported worldwide, were also identified in Chilean patients (one and two patients, respectively). This is the first report of T2 and T11 genotypes isolated from Acanthamoeba keratitis patients in South America. It is also the first report of the T2 genotype circulating in this continent. Analysis of the diagnostic fragment 3 region of the 18S rRNA gene showed 24 T4 variants, with a predominance of the sub-genotype T4/A, followed by T4/B, T4/G, T4/C and T4/D. Bayesian analysis revealed three groups among the T4 variants: two well supported groups that included 12 and 7 sub-genotypes, respectively, and a weakly supported group that included 5 sub-genotypes. Most of the predominant T4 sub-genotypes belonged to the same group, which included 71.3% of the patients, while some minority variants lied mainly in the other two clusters.ConclusionsT2, T4 and T11 genotypes were predominantly isolated from the Acanthamoeba keratitis patients in Chile. Chilean predominant T4 sub-genotypes, which have also been reported worldwide, formed a separate cluster of the minority T4 variants. This study provides useful information about the predominant genotypes and subgenotypes that would be useful in selecting suitable strains to develop immunological and/or molecular diagnostic assays in Chile.

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Detection of Acanthamoeba spp. using carboxylesterase antibody and its usage for diagnosing Acanthamoeba-keratitis.

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