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Molecular diagnosis of Acanthamoeba keratitis: evaluation in rat model and application in suspected human cases
Adriana Oliveira Costa1, Cinthia Furst2, Lucas Oliveira Rocha3
1Departamento 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.
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.
Insights
Rapid diagnosis of Acanthamoeba keratitis (AK) is crucial. A magnetic bead DNA extraction kit combined with semi-nested PCR (snPCR) offers a highly sensitive and effective method for detecting Acanthamoeba in corneal samples.
Area of Science:
- Ophthalmology
- Infectious Diseases
- Molecular Biology
Background:
- Acanthamoeba keratitis (AK) is a severe corneal infection leading to visual impairment.
- Accurate and rapid diagnostic methods are essential for effective AK treatment.
- Current diagnostic techniques may lack sufficient sensitivity and speed.
Purpose of the Study:
- To evaluate and compare two DNA extraction techniques for Acanthamoeba detection.
- To develop and validate a semi-nested PCR (snPCR) assay for Acanthamoeba diagnosis.
- To assess the efficacy of the optimized molecular method in an animal model and human cases.
Main Methods:
- Comparison of magnetic bead-based DNA extraction with alkaline lysis.
- Development of a semi-nested PCR (snPCR) targeting the 18S rRNA gene for Acanthamoeba.
- Validation using cultured Acanthamoeba, corneal scrapings/biopsies from an AK rat model, and human clinical samples.
Main Results:
- Magnetic bead DNA extraction was more efficient than alkaline lysis, detecting single Acanthamoeba cells.
- The optimized snPCR and DNA extraction method achieved 100% positivity in experimentally infected rat corneal scrapings.
- The molecular method diagnosed 80% of human AK cases, compared to 40% by culture.
Conclusions:
- An animal model is effective for validating diagnostic tools for Acanthamoeba keratitis.
- The combination of magnetic bead DNA extraction and snPCR provides a sensitive and effective diagnostic approach for AK.
- This optimized molecular protocol offers a significant improvement over traditional culture methods for diagnosing Acanthamoeba keratitis.
