ABSTRACT
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.
ABSTRACT
PURPOSE: To assess the level of stereopsis in school children with spectacle-corrected refractive errors using Titmus fly and Randot stereo tests, evaluate the factors associated with the level of stereopsis, and determine the level of agreement between the two tests. METHODS: A cross-sectional study was done on 5- to 18-year-old school-children wearing spectacles for at least 1-month duration. Visual acuity was assessed using Snellen's visual acuity chart, and their spectacle correction being used currently was measured using an auto lensmeter. The level of stereopsis was assessed using Randot and Titmus fly stereo tests. Data were entered using Microsoft Excel and analyzed using IBM-SPSS version 20, Chicago, IL. The associations between stereopsis and type of refractive error, visual acuity, age, and gender were analyzed. An agreement between Randot and Titmus fly test was done using Kappa statistics. RESULTS: A total of 222 children (101 boys and 121 girls; mean age 13 years) were assessed. Astigmatism was the most prevalent refractive error (60.4%), followed by myopia (24.8%) and hypermetropia (1.4%). Thirty children (13.5%) had anisometropia. All hyperopes had normal stereopsis. Children with spherical myopia had better stereopsis, followed by astigmatism and anisometropia in the same order (P = 0.036). Children with anisometropia ≤1.5 D had better stereopsis than anisometropia more than 1.5 D. Stereopsis was also found to have no correlation with the age and visual acuity at the time of testing or the age at which the child first started wearing spectacles. Stereopsis values obtained from Randot and Titmus fly stereo tests showed moderate agreement with Kappa value 0.581. CONCLUSION: Anisometropia and astigmatism are the most critical factors determining the level of stereopsis in refractive errors.
