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Rapid and Specific Detection of Acinetobacter baumannii Infections Using a Recombinase Polymerase Amplification/Cas12a-based System
Published on: April 25, 2025
Development and Clinical Evaluation of a CRISPR/Cas12a-Based Nucleic Acid Detection Platform for the Diagnosis of
Hanith Raj Deivarajan1, Vignesh Elamurugan1,2, Padmapriya Sivashanmugam1,3
1Department of Microbiology, Aravind Medical Research Foundation, Madurai, Tamil Nadu, India.
Objective:
The objective of this study was to develop a rapid and accurate clustered regularly interspaced short palindromic repeats (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 3 tertiary care institutions in South India.
Methods:
The RID-MyC assay utilized recombinase polymerase amplification targeting the 18S ribosomal RNA gene for isothermal amplification, followed by a CRISPR/Cas12a reaction. This was benchmarked against microscopy, culture, and polymerase chain reaction 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 4 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% confidence interval [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).
Conclusions:
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.
Financial Disclosures:
Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.
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