Abstract
Advances in genomics have the potential to revolutionize clinical diagnostics. Here, we examine the microbiome of vitreous (intraocular body fluid) from patients who developed endophthalmitis following cataract surgery or intravitreal injection. Endophthalmitis is an inflammation of the intraocular cavity and can lead to a permanent loss of vision. As controls, we included vitreous from endophthalmitis-negative patients, balanced salt solution used during vitrectomy and DNA extraction blanks. We compared two DNA isolation procedures and found that an ultraclean production of reagents appeared to reduce background DNA in these low microbial biomass samples. We created a curated microbial genome database (>5700 genomes) and designed a metagenomics workflow with filtering steps to reduce DNA sequences originating from: (i) human hosts, (ii) ambiguousness/contaminants in public microbial reference genomes and (iii) the environment. Our metagenomic read classification revealed in nearly all cases the same microorganism that was determined in cultivation- and mass spectrometry-based analyses. For some patients, we identified the sequence type of the microorganism and antibiotic resistance genes through analyses of whole genome sequence (WGS) assemblies of isolates and metagenomic assemblies. Together, we conclude that genomics-based analyses of human ocular body fluid specimens can provide actionable information relevant to infectious disease management.
Highlights
Cataract is a condition in which the lens of the eye becomes progressively opaque and is one of the major causes of reversible visual loss
To evaluate the use of shotgun metagenomic sequencing for the identification of potential disease-causing agents in postoperative endophthalmitis, we collected vitreous during vitrectomy from 14 patients with endophthalmitis (7 post cataract surgery, 7 post intravitreal injection) (Fig. 1, Supplementary Table S1)
As there exist no standard procedure for the isolation of DNA from vitreous, we examined two procedures using the QIAamp DNA Mini Kit (QIA) and QIAamp UCP Pathogen Mini kit (UCP) and 4 extraction controls were included per kit (Fig. 1)
Summary
Cataract is a condition in which the lens of the eye becomes progressively opaque and is one of the major causes of reversible visual loss. A vitrectomy is a procedure in which the vitreous body of the eye, which is the immobile gel-like fluid that occupies the space between the lens and retina, is aspirated and replaced by balanced salt solution. A vitreous tap is a more simple procedure where the vitreous is aspirated without being replaced by balanced salt solution In both procedures, antibiotics, such as vancomycin combined with ceftazidime, are being injected into the vitreous body to treat the infection. Polymerase chain reaction can increase the rate of identifying the microorganisms by 20%12, but in many endophthalmitis cases a causative agent cannot be identified. It is unclear, whether the vitreous in endophthalmitis may contain multiple microorganisms that are not all being detected with the current methods. Non-infectious endophthalmitis can present as a variant of TASS (toxic anterior segment syndrome) and these patients may benefit from steroid instead of antibiotic treatment to obtain a better visual outcome[13]
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