Comprehensive pathogen detection for ocular infections.

Area of Science:

• Ophthalmology
• Infectious Diseases
• Molecular Diagnostics

Background:

• Pathogen-directed PCR has been a cornerstone of ocular infection diagnostics since the late 1990s.
• While effective for low biomass samples, PCR's limited diagnostic range necessitates alternative molecular methods for comprehensive ocular infection testing.

Purpose of the Study:

• To evaluate the performance of high-throughput RNA sequencing (RNA-seq) for detecting infectious agents in ocular samples.
• To compare RNA-seq's diagnostic capabilities against established pathogen-directed PCR assays in patients with suspected ocular infections.

Main Methods:

• A comparative study was conducted using remnant nucleic acids from 41 aqueous or vitreous samples.
• RNA-seq was performed in a masked fashion and compared with pathogen-directed PCR results.
• Orthogonal testing was employed to validate discordant results between the two methods.

Main Results:

• RNA-seq demonstrated 100% positive percent agreement and 92.6% negative percent agreement compared to PCR.
• RNA-seq identified unexpected pathogens in 9.7% of samples, expanding the diagnostic scope.
• Orthogonal testing confirmed two pathogens initially detected exclusively by RNA-seq.

Conclusions:

• RNA-seq accurately identifies both common and rare pathogens in ocular fluid samples.
• This unbiased molecular approach holds significant potential for enhancing the diagnosis of ocular infections.
• Further clinical utility studies are warranted to fully integrate RNA-seq into routine practice.