NGS ANALYSIS OF CORNEAL PATHOGENS: CAN WE USE IT IN A CLINICAL SETTING?

Abstract

Purpose : To test the utilization of next generation DNA sequencing (NGS) techniques for comprehensive analysis of the pathogenic microbiome on the human ocular surface.

Methods : The study was approved by the University of Miami IRB. Two complementary NGS strategies were used to study the ocular surface microbiome: 1) amplification-based sequencing of 16S ribosomal RNA gene libraries and 2) direct metagenomics sequencing (MS) using Illumina HiSeq platform. 16S provided genus-level phylogenetic classification of bacterial DNA reads; MS allowed identification of DNA of bacterial, fungal, viral and protozoan origin. Bioinformatics for the 16S data was utilized using the RDPII database and a TUIT classifier; the MS data was performed using the Cosmos ID curated database. We used a pathogenicity and virulence factor profiling to calculate the risk of keratitis development as a result of homeostatic microflora disruption. P-values were calculated using the HMP tree analysis with a 95% confidence interval.

Results : A total of 75 healthy and 6 ulcerative keratitis subjects were recruited and had their corneal epithelial samples analyzed for bacterial composition using NGS. Our 16S sequencing results showed dramatic changes in the composition of the healthy ocular bacterial community at the onset of bacterial keratitis. The infected corneal biome showed severe dysbiosis with opportunistic species of Cloacibacterium, Acinetobacter and Novosphingobium strongly associated with the principal pathogen Pseudomonas aeruginosa. The application of MS allowed us to detect fungal and viral components in addition to bacterial species, perform resistance and virulence profiling, and reduce 16S amplification-induced errors introduced by variability in 16S rRNA gene copy number and false positives. A robust bioinformatics and statistical pipeline for analysis of 16S and metagenomics data allowed for integration of NGS microbiome analysis into clinical practice. The 16S rRNA and deep metagenomic sequencing strategies revealed low density, but high bacterial diversity on the human conjunctiva and cornea, demonstrating a polymicrobial nature of ocular infections.

Conclusions : Our results provide a proof of consent for a clinical prospective trial to use the NGS-based pathogen diagnostics as a new standard of care in a head to head comparison against current microbiological culture-based techniques.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.