An All-in-One Highly Multiplexed Diagnostic Assay for Rapid, Sensitive, and Comprehensive Detection of Intraocular Pathogens

center dot PURPOSE: Intraocular infections are sight-threatening conditions that can lead to vision loss. Rapid identifica-tion of the etiologies plays a key role in early initiation of effective therapy to save vision. However, current di-agnostic modalities are time consuming and lack sensi-tivity and inclusiveness. We present here a newly devel-oped comprehensive ocular panel designed to improve di-agnostic yields and provide a tool for rapid and sensitive pathogen detection.center dot DESIGN: Experimental laboratory investigation.center dot METHODS: A panel containing 46 pathogens and 2 re-sistance/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 Pro-filer. 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.center dot RESULTS: Analytical studies demonstrated highly sen-sitive detection of a broad spectrum of pathogens, includ-ing bacteria, viruses, and parasites, with limits of detec-tion being as low as 2.5 femtograms per reaction. We also found excellent target specificity, with minimal cross-reactivity detected. The custom-designed NanoString oc-ular panel correctly identified the causative agent from all clinical specimens positive for a variety of pathogens.center dot 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 in-traocular infections. (Am J Ophthalmol 2023;250: 82-94.(c) 2023 Elsevier Inc. All rights reserved.)

Automated summary

A newly developed ocular panel was designed to improve the diagnostic yield of intraocular infections by rapidly and sensitively detecting a wide range of pathogens.