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.

The first Acanthamoeba keratitis case in the Midwest region of Brazil: diagnosis, genotyping of the parasite and disease outcome

“…In an experimental study conducted in Brazil on corneal scraping and biopsies from an AK rat model, the authors evaluated two DNA extraction techniques and a seminested-PCR (snPCR) targeting the 18S rRNA gene to detect Acanthamoeba cysts and trophozoites. Similar to our fi ndings, they reported higher positivity when comparing culture and PCR methods, thus demonstrating the importance of performing two diagnostic techniques for human keratitis 7 .…”

Development, Optimization, and Validation of a Multiplex Real-Time PCR Assay on the BD MAX Platform for Routine Diagnosis of Acanthamoeba Keratitis

“…Several other protocols for cellwall disruption have been described, including beadextraction methods, which also seem to be an efficient way of optimizing DNA extraction. 22 Molecular biology platforms are continually being adapted to the needs of users. Ready-to-use kits and random-access platforms are currently being expanded in microbiology laboratories.…”

The acantamoeba lesions of the cornea (diagnosis) (Review of literature)

“…Использование количественной ПЦР в реальном времени не требует гель-электрофореза, сокращает трудоемкость обработки, а также позволяет повысить чувствительность более 89,3% [49]. М. Kandori et al разработали мультиплексный qПЦР-метод, чтобы обнаружить различные роды АА [50].…”