Diagnostic Accuracy Of A Crispr-Based Assay In Smear- And Culture-Negative Fungal Keratitis

To determine the diagnostic accuracy of a CRISPR-based Rapid Identification of Mycoses using CRISPR (RID-MyC) assay for detecting FK in patients with negative smear and culture results, using in vivo confocal microscopy (IVCM) as the reference standard.

Design: Prospective diagnostic accuracy study conducted from December 2024 to March 2025.

Setting: Single-center, tertiary ophthalmology referral hospital

Of 245 consecutive patients clinically suspected to have microbial keratitis, 82 were smear-negative. After exclusions due to contraindications or positive subsequent cultures, 41 smear- and culture-negative patients were ultimately included in the final analysis.  All included participants underwent corneal scraping for RID-MyC assay and imaging by IVCM.  The primary outcomes were sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and diagnostic concordance of the RID-MyC assay compared with IVCM results.

Of the 41 smear- and culture-negative patients (mean [SD] age, 51.0 [14.6] years; 20 [48.7%] men, 21 [51.2%] women), RID-MyC demonstrated sensitivity of 82.1% (95% CI, 63%-94%) and specificity of 76.9% (95% CI, 46%-95%). Positive predictive value was 88.5% (95% CI, 74%-95%), and negative predictive value was 66.7% (95% CI, 46%-82%). Concordance between RID-MyC and IVCM was observed in 33 cases (80.5%). Notably, prior antifungal treatment was most frequent (4 of 5 [80%]) among cases with positive IVCM but negative RID-MyC results. Conversely, all cases (3 of 3 [100%]) with negative IVCM but positive RID-MyC findings had smaller, peripheral or paracentral lesions.

In patients with smear- and culture-negative FK, RID-MyC assay showed good diagnostic accuracy comparable with IVCM and was feasible in all cases, including those in whom imaging was not possible. With its rapid turnaround and minimal equipment needs, RID-MyC may serve as a practical adjunct to conventional diagnostics, particularly in high-burden, resource-limited settings where IVCM is unavailable or contraindicated.