Abstract
Purpose :
This study evaluates the RID-MyC (Rapid Identification of Mycoses using CRISPR), a novel CRISPR-based assay against traditional methods like culture, PCR, and NGS for diagnosing Fungal Endophthalmitis (FE), a major cause of blindness, especially in tropical regions. Current diagnostic approaches often fall short, with culture methods yielding limited results and PCR's effectiveness reduced by intrinsic inhibitors in intraocular fluids. NGS data indicates that many 'culture-negative' FE cases might actually be fungal, highlighting the need for improved diagnostics. The RID-MyC assay's efficacy in accurately identifying FE is thus critically assessed.
Methods :
Methods: The study analyzed 119 intraocular specimens from 96 patients suspected of having infective endophthalmitis and 10 control patients using the RID-MyC assay, microscopy, culture, and PCR. Specimens were divided into two aliquots post-collection. One aliquot was used for microbiological identification with Gram stain and culture on blood agar, chocolate agar, thioglycolate broth, brain heart infusion broth, and potato dextrose agar within 30 minutes of collection. The other aliquot was stored at -20°C for DNA extraction. The RID-MyC assay involved recombinase polymerase amplification targeting the 18S rRNA gene, followed by CRISPR/Cas12a reaction. A result was considered positive if it met or exceeded a cut-off threshold based on the negative control samples. Additionally, 36 specimens were selected for targeted 16S and ITS ribosomal RNA sequencing.
Results :
None of the 10 control samples tested positive for fungal endophthalmitis (FE) using culture, PCR, or RID-MyC. Among 109 specimens from suspected patients, detection rates were 9% for culture, 13.8% for PCR, and 23.8% for RID-MyC. Concordance rates were 42% between RID-MyC and PCR, and 87% with NGS. Median time for a positive RID-MyC result was 61.5 minutes, varying by specimen type but not significantly. Endogenous endophthalmitis cases took longer to diagnose (73.75 minutes) compared to trauma-induced (59.7 minutes) and postoperative cases (52.25 minutes), with a significant association found in multivariate analysis for longer diagnosis time in endogenous cases.
Conclusions :
The rapidity, superior sensitivity, specificity and cost-effectiveness of the CRISPR-based RID-MyC assay make it a valuable tool for diagnosing fungal endophthalmitis.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.