Rapid one-tube RPA-coupled CRISPR/Cas12a-based RID-MyC assay for the diagnosis of fungal keratitis.

PURPOSE: This study introduces and evaluates the single-tube rapid identification of mycoses using CRISPR (ST-RID-MyC) assay. This novel diagnostic tool combines recombinase polymerase amplification (RPA) with CRISPR/Cas12a for the rapid and precise diagnosis of fungal keratitis (FK).

DESIGN

Prospective cross-sectional study.

METHODS

Corneal scrapings from 61 patients with suspected microbial keratitis were collected at the Cornea Department of a Tertiary Eye Care Center. The study assessed the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the ST-RID-MyC assay. Additional measures included concordance rates with traditional diagnostic methods and the time to diagnosis.

RESULTS

The ST-RID-MyC assay exhibited a sensitivity of 90% and a specificity of 90.48%, with a PPV of 94.74% and an NPV of 82.61%. The ST-RID-MyC showed substantial agreement with culture and microscopy and perfect concordance with conventional RID-MyC. The mean time to diagnosis was significantly reduced (P < 0.001) using the ST-RID-MyC assay, compared to the traditional RID-MyC assay (6 vs. 32 minutes). Visual assessments demonstrated a high level of inter-observer agreement (kappa = 0.832).

CONCLUSIONS

The ST-RID-MyC assay, combining RPA and CRISPR/Cas12a in a single-tube system, offers a rapid, accurate, and resource-efficient diagnostic method for FK, potentially transforming clinical management of this condition by enabling faster therapeutic decisions.