@article {1664986,
	title = {An All-in-One Highly Multiplexed Diagnostic Assay for Rapid, Sensitive, and Comprehensive Detection of Intraocular Pathogens},
	journal = {Am J Ophthalmol},
	volume = {250},
	year = {2023},
	month = {2023 Jun},
	pages = {82-94},
	abstract = {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\&nbsp;=\&nbsp;15) and endophthalmitis (n\&nbsp;=\&nbsp;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.},
	keywords = {Bacteria, Endophthalmitis, Eye Infections, Humans, Polymerase Chain Reaction, Sensitivity and Specificity},
	issn = {1879-1891},
	doi = {10.1016/j.ajo.2023.01.021},
	author = {Bispo, Paulo J M and Belanger, Nicole and Li, Ashley and Liu, Renee and Susarla, Gayatri and Chan, Weilin and Chodosh, James and Gilmore, Michael S and Sobrin, Lucia}
}
