Development and Clinical Evaluation of a CRISPR/Cas12a-based Nucleic Acid Detection Platform for the Diagnosis of Keratomycoses

Objective The objective of this study was to develop a rapid and accurate CRISPR/Cas12a-based molecular diagnostic assay (Rapid Identification of Mycoses using CRISPR, RID-MyC Assay) to detect fungal nucleic acids and to compare it with existing conventional mycologic methods for the diagnosis of fungal keratitis (FK). Design This study was structured as a development and validation study focusing on the creation and assessment of the RID-MyC assay as a novel diagnostic modality for FK. Subjects Participants comprised 142 individuals presenting with suspected microbial keratitis at three tertiary care institutions in South India. Methods The RID-MyC assay utilized recombinase polymerase amplification (RPA) targeting the 18S rRNA gene for isothermal amplification, followed by a CRISPR/Cas12a reaction. This was benchmarked against microscopy, culture, and polymerase chain reaction (PCR) for the diagnosis of FK. Main Outcome Measures The primary outcome measures focused on the analytical sensitivity and specificity of the RID-MyC assay in detecting fungal nucleic acids. Secondary outcomes measured the assay's diagnostic sensitivity and specificity for FK, including its concordance with conventional diagnostic methods. Results The RID-MyC assay exhibited a detection limit ranging from 13.3 to 16.6 genomic copies across four common fungal species. In patients with microbial keratitis, the RID-MyC assay showed substantial agreement with microscopy (kappa = 0.714) and fair agreement with culture (kappa = 0.399). The assay demonstrated a sensitivity of 93.27% (95% CI, 86.62% - 97.25%) and a specificity of 89.47 % (95% CI, 66.86 % - 98.70 %) for FK diagnosis, with a median diagnostic time of 50 minutes (range, 35 - 124 minutes). Conclusion The RID-MyC assay, utilizing CRISPR-Cas12a technology, offers high diagnostic accuracy for FK. Its potential for point-of-care use could expedite and enhance the precision of fungal diagnostics, presenting a promising solution to current diagnostic challenges.