Nested PCR as a superior diagnostic method for Acanthamoeba keratitis compared to conventional techniques.

Area of Science:

• Ophthalmology
• Medical Microbiology
• Molecular Diagnostics

Background:

• Acanthamoeba keratitis (AK) is a severe ocular infection leading to vision loss, particularly in contact lens wearers.
• Timely and precise diagnosis is critical for effective AK management and visual outcome preservation.
• Conventional diagnostic methods like culture exhibit limitations in speed and sensitivity for AK detection.

Purpose of the Study:

• To evaluate the diagnostic performance of nested polymerase chain reaction (PCR) targeting the 18S rRNA gene for Acanthamoeba keratitis (AK).
• To compare nested PCR against one-step PCR, culture, and in vivo confocal microscopy (IVCM) in suspected AK cases.
• To determine the optimal diagnostic strategy for rapid and accurate AK identification.

Main Methods:

• A prospective study involving 42 patients with suspected AK.
• Corneal scrapings were analyzed using in vivo confocal microscopy (IVCM), bacterial culture, one-step PCR, and nested PCR.
• Diagnostic accuracy metrics including sensitivity, specificity, and predictive values were calculated for each method.

Main Results:

• Nested PCR demonstrated the highest sensitivity (96.3%) for AK detection among all tested methods.
• All methods showed 100% specificity and positive predictive value.
• Nested PCR exhibited the highest negative predictive value (93.75%) and strong agreement with IVCM findings.

Conclusions:

• Nested PCR significantly enhances the sensitivity of Acanthamoeba detection in corneal samples, especially with low parasite loads.
• The combination of nested PCR and IVCM provides the most effective diagnostic approach for AK.
• Nested PCR is a reliable alternative diagnostic tool for AK when IVCM is unavailable or other methods are negative.