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Abstract：
UNASSIGNED: 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).
UNASSIGNED: 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.
UNASSIGNED: Participants comprised 142 individuals presenting with suspected microbial keratitis at 3 tertiary care institutions in South India.
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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).
UNASSIGNED: 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.
UNASSIGNED: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.
UNASSIGNED: 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.
UNASSIGNED: Participants comprised 142 individuals presenting with suspected microbial keratitis at 3 tertiary care institutions in South India.
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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).
UNASSIGNED: 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.
UNASSIGNED: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.
摘要：
■这项研究的目的是开发一种快速，准确的成簇的规则间隔短回文重复序列（CRISPR）/Cas12a的基于分子诊断方法（使用CRISPR快速鉴定真菌病，RID-MyC测定）以检测真菌核酸，并将其与现有的常规真菌学方法进行比较，以诊断真菌性角膜炎（FK）。
■本研究是一项开发和验证研究，重点是创建和评估RID-MyC测定作为FK的新型诊断方式。
■参与者包括在印度南部的3个三级护理机构中出现疑似微生物性角膜炎的142个人。
■RID-MyC分析利用靶向18S核糖体RNA基因的重组酶聚合酶扩增进行等温扩增，随后进行CRISPR/Cas12a反应。这是以显微镜为基准的，文化,和聚合酶链反应诊断FK。
■主要结果测量集中在RID-MyC测定在检测真菌核酸中的分析灵敏度和特异性。次要结果测量了测定对FK的诊断灵敏度和特异性，包括其与传统诊断方法的一致性。
■RID-MyC测定在4种常见真菌物种中表现出13.3至16.6个基因组拷贝的检测限。在微生物性角膜炎患者中，RID-MyC分析显示与显微镜检查基本一致(kappa=0.714),与培养物一致(kappa=0.399).该测定法显示出93.27%的灵敏度(95%置信区间[CI]，86.62%-97.25%），FK诊断的特异性为89.47%（95%CI，66.86%-98.70%），中位诊断时间为50分钟(范围，35-124分钟）。
■RID-MyC分析，利用CRISPR-Cas12a技术，为FK提供高诊断精度。其潜在的护理点使用可以加快和提高真菌诊断的准确性，为当前的诊断挑战提出了一个有希望的解决方案。
■专有或商业披露可在本文末尾的脚注和披露中找到。
■本研究是一项开发和验证研究，重点是创建和评估RID-MyC测定作为FK的新型诊断方式。
■参与者包括在印度南部的3个三级护理机构中出现疑似微生物性角膜炎的142个人。
■RID-MyC分析利用靶向18S核糖体RNA基因的重组酶聚合酶扩增进行等温扩增，随后进行CRISPR/Cas12a反应。这是以显微镜为基准的，文化,和聚合酶链反应诊断FK。
■主要结果测量集中在RID-MyC测定在检测真菌核酸中的分析灵敏度和特异性。次要结果测量了测定对FK的诊断灵敏度和特异性，包括其与传统诊断方法的一致性。
■RID-MyC测定在4种常见真菌物种中表现出13.3至16.6个基因组拷贝的检测限。在微生物性角膜炎患者中，RID-MyC分析显示与显微镜检查基本一致(kappa=0.714),与培养物一致(kappa=0.399).该测定法显示出93.27%的灵敏度(95%置信区间[CI]，86.62%-97.25%），FK诊断的特异性为89.47%（95%CI，66.86%-98.70%），中位诊断时间为50分钟(范围，35-124分钟）。
■RID-MyC分析，利用CRISPR-Cas12a技术，为FK提供高诊断精度。其潜在的护理点使用可以加快和提高真菌诊断的准确性，为当前的诊断挑战提出了一个有希望的解决方案。
■专有或商业披露可在本文末尾的脚注和披露中找到。
