一种一体化的高度多重诊断检测方法，用于快速、敏感、全面地检测眼内病原体。
An All-in-One Highly Multiplexed Diagnostic Assay for Rapid, Sensitive, and Comprehensive Detection of Intraocular Pathogens.
机构信息
From the Department of Ophthalmology (P.J.M.B., N.B., A.L., R.L., G.S., W.C., J.C., M.S.G., L.S.), Massachusetts Eye and Ear, Harvard Medical School, Boston, Massachusetts, USA; Infectious Disease Institute (P.J.M.B., N.B., J.C., M.S.G., L.S.), Harvard Medical School, Boston, Massachusetts, USA.
From the Department of Ophthalmology (P.J.M.B., N.B., A.L., R.L., G.S., W.C., J.C., M.S.G., L.S.), Massachusetts Eye and Ear, Harvard Medical School, Boston, Massachusetts, USA; Infectious Disease Institute (P.J.M.B., N.B., J.C., M.S.G., L.S.), Harvard Medical School, Boston, Massachusetts, USA.
出版信息
Am J Ophthalmol. 2023 Jun;250:82-94. doi: 10.1016/j.ajo.2023.01.021. Epub 2023 Jan 26.
PURPOSE
Intraocular infections are sight-threatening conditions that can lead to vision loss. Rapid identification of the etiologies plays a key role in early initiation of effective therapy to save vision. However, current diagnostic modalities are time consuming and lack sensitivity and inclusiveness. We present here a newly developed comprehensive ocular panel designed to improve diagnostic yields and provide a tool for rapid and sensitive pathogen detection.
DESIGN
Experimental laboratory investigation.
METHODS
A panel containing 46 pathogens and 2 resistance/virulence markers that are commonly detected in intraocular infections was developed. Genomic targets were scrutinized for stretches predicted to be specific for a particular species while being conserved across different strains. A set of primers for sample enrichment, and two 50mer NanoString compatible probes were then designed for each target. Probe-target hybrids were detected and quantified using the NanoString nCounter SPRINT Profiler. Diagnostic feasibility was assessed in a pilot clinical study testing samples from infectious retinitis (n = 15) and endophthalmitis (n = 12) patients, for which the etiologies were confirmed by polymerase chain reaction (PCR) or culture.
RESULTS
Analytical studies demonstrated highly sensitive detection of a broad spectrum of pathogens, including bacteria, viruses, and parasites, with limits of detection being as low as 2.5 femtograms per reaction. We also found excellent target specificity, with minimal cross-reactivity detected. The custom-designed NanoString ocular panel correctly identified the causative agent from all clinical specimens positive for a variety of pathogens.
CONCLUSION
This highly multiplexed panel for pathogen detection offers a sensitive, comprehensive, and uniform assay run directly on ocular fluids that could significantly improve diagnostics of sight-threatening intraocular infections.
目的
眼内感染可导致视力丧失，是威胁视力的疾病。快速确定病因对于尽早开始有效治疗以挽救视力起着关键作用。然而，目前的诊断方法耗时且缺乏敏感性和包容性。我们在此介绍一种新开发的综合眼部检测面板，旨在提高诊断效果，并提供一种快速、敏感的病原体检测工具。
设计
实验性实验室研究。
方法
开发了一个包含 46 种病原体和 2 种耐药/毒力标志物的面板，这些标志物通常在眼内感染中检测到。对基因组靶标进行了仔细检查，以寻找在不同菌株中保守但特定于特定物种的特定区域。然后为每个目标设计了一组用于样品富集的引物和两个 50mer NanoString 兼容探针。使用 NanoString nCounter SPRINT Profiler 检测和定量探针-靶标杂交物。在一项试点临床研究中评估了诊断可行性，该研究测试了来自传染性视网膜炎（n=15）和眼内炎（n=12）患者的样本，这些病因通过聚合酶链反应（PCR）或培养得到确认。
结果
分析研究表明，该广谱病原体检测面板具有高度灵敏性，可检测包括细菌、病毒和寄生虫在内的病原体，检测限低至 2.5 飞克/反应。我们还发现了出色的靶标特异性，检测到的交叉反应最小。该定制的 NanoString 眼部检测面板可正确识别出所有临床标本中各种病原体的致病因子。
结论
这种用于病原体检测的高度多重检测面板提供了一种灵敏、全面和统一的检测方法，可直接在眼液上进行，这可能会显著改善威胁视力的眼内感染的诊断。
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