A one-step multiplex PCR for acanthamoeba keratitis diagnosis and quality samples control

Area of Science:

• Ophthalmology
• Molecular Biology
• Parasitology

Background:

• Acanthamoeba spp. keratitis (AK) is an increasing cause of infectious keratitis.
• Accurate and timely diagnosis is crucial for effective treatment and visual outcomes.
• Existing diagnostic methods for AK can be limited, necessitating improved molecular techniques.

Purpose of the Study:

• To develop and evaluate a novel multiplex PCR technique for Acanthamoeba DNA detection.
• To incorporate human DNA detection for improved PCR result interpretation.
• To establish a sensitive and specific molecular diagnostic tool for AK.

Main Methods:

• Optimized DNA extraction procedures, including thermal-shock pretreatment.
• Developed a multiplex Acanthamoeba beta-globin PCR (MAB-PCR) assay.
• Evaluated analytical parameters, including specificity, sensitivity, and PCR inhibitors in eye drops.
• Tested MAB-PCR on a prospective series of 28 corneal scrapings.

Main Results:

• The optimized DNA extraction and MAB-PCR demonstrated high specificity and sensitivity, detecting as little as 0.02 Acanthamoeba genome.
• No tested eye drops acted as complete PCR inhibitors.
• Out of 28 corneal scrapings, 3 were true positives, and 18 were true negatives; 7 samples were uninterpretable due to insufficient material.
• Positive cases showed favorable progression with appropriate treatment.

Conclusions:

• MAB-PCR is a valuable new tool for the rapid diagnosis of AK.
• The assay allows for prompt treatment initiation, addressing an underestimated cause of infectious keratitis.
• MAB-PCR surpasses the gold standard in diagnosing AK and simultaneously assesses sample quality for reliable interpretation.