Clinical Utility of CRISPR-Based RID-MyC Assay in Smear and Culture-Negative Fungal Keratitis: A Case Series

To assess the clinical utility of the clustered regularly interspaced short palindromic repeats/Cas12a-based RID-MyC assay in diagnosing fungal keratitis (FK) in cases where conventional smear and culture methods fail to identify the causative pathogen.

This retrospective case series included 5 patients with clinically suspected FK and negative smear and culture results who were evaluated in the Cornea Department at Aravind Eye Hospital, Coimbatore, India, between January 1, 2024, and March 31, 2024. The primary outcome was the diagnostic performance of the RID-MyC assay in detecting fungal nucleic acids in cases of suspected FK with negative smear and culture results. In vivo confocal microscopy served as a reference standard to validate the RID-MyC assay findings.

The RID-MyC assay successfully detected fungal nucleic acids in 3 cases, corroborated by in vivo confocal microscopy findings suggestive of fungal filaments, leading to targeted antifungal therapy and resolution of the infections. In addition, 2 cases tested negative for fungal nucleic acids, aligning with clinical and confocal evidence of nonfungal etiology, thus guiding appropriate alternative treatments that led to clinical improvement.

The RID-MyC assay demonstrates clinical utility in diagnosing FK in scenarios where conventional smear and culture methods prove inadequate, such as in cases with prior antifungal therapy or polymicrobial infections. This assay facilitates accurate diagnosis and timely initiation of appropriate antifungal treatment without the need for sophisticated equipment or expertise, making it particularly valuable in resource-limited settings.