Ocular Microbiology and Immunology Group
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2026 OMIG Abstract

Improving the Diagnosis of Culture-Negative Bacterial Endophthalmitis Using Targeted Next-Generation Sequencing: Illumina Versus Nanopore Platform Comparison

Brionna L. Bennett, Lucia Sobrin, Paulo J.M. Bispo, and K. Matthew McKay

Department of Ophthalmology, Massachusetts Eye and Ear / Harvard Medical School,
Boston, Massachusetts

Purpose: Endophthalmitis is a serious vision-threatening intraocular infection requiring prompt pathogen identification. Conventional culture-based diagnostics yield negative results in 60–70% of suspected cases, leaving clinicians reliant on empiric broad-spectrum antimicrobials. This study evaluates the diagnostic yield of targeted Illumina 16S rRNA next-generation sequencing (NGS) in culture-negative endophthalmitis and presents the first head-to-head platform comparison with Oxford Nanopore Technologies (ONT) full-length 16S sequencing in ophthalmic diagnostics.

Methods: A total of 124 culture-negative intraocular specimens (aqueous and vitreous humor) from 101 unique patients were analyzed using platform-specific 16S sequencing workflows. Illumina data were processed with Mothur/GreenGenes targeting the V1–V2 hypervariable region; Nanopore data were processed with EPI2ME/NCBI databases using full-length 16S amplicons on the GridION platform. A formally derived blank-based contamination removal framework was applied independently per platform, establishing a noise floor of 15.2% for Illumina and 9.9% for Nanopore from platform-specific negative extraction controls. Platform-level concordance was assessed among 103 paired specimens with sequencing results on both platforms. Statistical analyses were performed using IBM SPSS Statistics Version 28 with Wilson score 95% confidence intervals and Cohen’s kappa.

Results: Illumina 16S NGS identified clinically relevant bacterial pathogens in 38 of 124 specimens (30.6%; 95% CI 23.2-39.2%), with a median dominant taxon relative abundance of 32.9% (IQR 21.3-77.1%). Detected organisms were predominantly fastidious species underrepresented in conventional culture, including Staphylococcus epidermidis (26.3%), Haemophilus influenzae (23.7%), and Streptococcus spp. (21.1%). Among 103 paired specimens, Nanopore identified 34 sequencing-positive cases (33.0%; 95% CI 24.7-42.6%), with results available within approximately 29 hours of sample receipt. Organism-level concordance among the 25 specimens called sequencing-positive on both platforms was 100% following taxonomy harmonization, with zero confirmed discordant organism-level calls (κ = 0.604; p < .001).

Conclusions: Targeted 16S NGS identified clinically relevant pathogens in approximately one-third of culture-negative endophthalmitis specimens. A formally derived, platform-specific blank-based contamination removal framework, provides a reproducible, control-anchored approach to positivity thresholding in low-biomass ocular specimens. Illumina and Nanopore demonstrated 100% organism-level concordance when both platforms independently called positive, with Nanopore offering marginally higher sensitivity and a turnaround time of 29 hours from sample receipt.