Abstract
Purpose :
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 and PCR for the diagnosis of Fungal Keratitis and Fungal Endophthalmitis.
Methods :
Isothermal amplification was performed using recombinase polymerase amplification targeting the 18S rRNA gene followed by the CRISPR/Cas12a reaction. Development and validation of the RID-MyC assay were performed using contrived reference strains and clinical isolates. The results of the RID-MyC assay were compared to those obtained by PCR in patients with suspected microbial keratitis and endophthalmitis. Corneal swabs and scrapes collected from 75 consecutive cases of clinically suspected microbial keratitis and 96 intraocular specimens collected from 75 consecutive patients with clinically suspected infective endophthalmitis and 10 control patients were analyzed prospectively.
Results :
Of the 75 corneal scrapes, 72 (96%) were positive for fungus by standard mycologic methods of which 55 (73.3%) were positive by both microscopy and culture and 17 (22.6%%) were positive only by microscopy. The sensitivity, specificity, PPV, and NPV of RID-MyC were 94.4%, 100%, 100 %, and 42.9%. There was no significant difference between the sensitivities and specificities of PCR and RID-MyC (p=0.157).
Among the 86 intraocular specimens from patients with suspected endophthalmitis, none were positive for fungus by conventional mycological examinations. Eighteen were positive for fungus by RID-MyC of which 13 were positive also by panfungal PCR. The concordance rate between PCR and RID-MyC was 93%. The clinical course review and next-generation sequencing indicated that the 5 discordant samples (RID-MyC positive but PCR negative) were from patients with fungal endophthalmitis.
Conclusions :
Here, we combined isothermal amplification and CRISPR-Cas12a to develop a rapid (45 – 60 min) and accurate assay for the diagnosis of fungal keratitis and endophthalmitis. This is the first study to describe a CRISPR-based assay for the broad-range detection of fungal nucleic acids in any system and also the first study to describe a CRISPR-based assay for the diagnosis of ophthalmic infections.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.