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

• Published in: American journal of ophthalmology Vol. 250; pp. 82 - 94
• Main Authors: Bispo, Paulo J.M., Belanger, Nicole, Li, Ashley, Liu, Renee, Susarla, Gayatri, Chan, Weilin, Chodosh, James, Gilmore, Michael S., Sobrin, Lucia
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-06-2023
• Subjects: Bacteria - genetics; Endophthalmitis - diagnosis; Eye Infections; Humans; Polymerase Chain Reaction; Sensitivity and Specificity
• ISSN: 0002-9394; 1879-1891
• DOI: 10.1016/j.ajo.2023.01.021

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.
Experimental laboratory investigation.
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.
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.
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.