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

Acanthamoeba keratitis (AK) is a severe eye infection that can cause severe vision loss, especially in contact lens wearers. Rapid and accurate diagnosis is crucial for achieving favorable outcomes. Traditional methods, such as culture, are slow and have variable sensitivity, while molecular techniques offer improved detection. This study evaluated nested polymerase chain reaction (PCR) targeting the 18S ribosomal RNA (rRNA) gene for rapid AK diagnosis, comparing its performance to one-step PCR, culture, and in vivo confocal microscopy (IVCM). This study included 42 suspected AK patients who underwent IVCM and corneal scraping for culture, PCR, and nested PCR. AK diagnosis was confirmed based on clinical findings, risk factors, treatment response, and at least one positive diagnostic test. Out of 42 patients, 27 were positive for Acanthamoeba with at least one of the diagnostic methods. Based on the diagnosis of definite AK, the sensitivity of IVCM, culture, PCR, and nested PCR was 77.78%, 37.04%, 62.96%, and 96.3%, respectively. The specificity and positive predictive values were 100% for all the tests. The negative predictive values of IVCM, culture, PCR, and nested PCR were 71.43%, 46.88%, 60%, and 93.75%, respectively. Nested PCR results displayed the highest agreement with IVCM. In conclusion, nested PCR enhances AK detection compared to conventional methods, improving sensitivity in low corneal scrape quantities. Combining high-sensitivity tests increases diagnostic accuracy, and nested PCR with IVCM is the most effective approach. When IVCM is unavailable, nested PCR serves as a reliable diagnostic tool, especially when culture and one-step PCR yield negative results.