Retrospective study of Acanthamoeba keratitis: Three-year experience with an integrated clinical and diagnostic workflow.

Acanthamoeba keratitis (AK) is a rare but sight-threatening corneal infection, caused by free-living protozoa of the genus Acanthamoeba. Although cases are rising in high-income countries, the nonspecific presentation and overlapping features with other keratitis frequently result in misdiagnosis of AK, with poorer outcomes and impaired quality of life. A delayed diagnosis can allow the infection to progress to deeper corneal layers, increasing the risk of severe structural damage and requiring emergency keratoplasty to preserve ocular integrity and regain vision. This retrospective study describes a three-year experience at a Northern Italy tertiary center using an integrated diagnostic workflow combining clinical evaluation, in vivo confocal microscopy, culture, and -since 2024- real-time PCR. Among 39 patients with suspected AK, 11 cases were confirmed. Culture showed 92 % sensitivity in first diagnoses but decreased to 77 % when relapses were included. PCR, introduced as a routine tool in 2024, showed 100 % sensitivity in first diagnoses and proved crucial in detecting low-burden or culture-negative relapses. Confocal microscopy reached a sensitivity and specificity of 82 %. Early targeted anti-amoebic therapy, guided by this integrated approach, contributed to favorable outcomes in most cases, limiting the need for surgical intervention. These findings support the utility of combining methods for timely and accurate diagnosis of AK. Despite ongoing challenges in the clinical management of AK, our findings reinforce the critical value of adopting molecular diagnostics to enable earlier, more accurate identification and targeted treatment.