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Liying Low, Radhika Pooja Patel, Ghazala Begum, Pablo Fuentes-Utrilla, Graham R Wallace, Philip Ian Murray, Saaeha Rauz; Nanopore Sequencing for Rapid Diagnosis of Microbial Keratitis. Invest. Ophthalmol. Vis. Sci. 2019;60(9):825.
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© ARVO (1962-2015); The Authors (2016-present)
Microbial keratitis is a leading cause of blindness worldwide. Prognosis is dependent on rapid pathogen identification and early treatment. In this study, we aim to investigate and optimise the use of a novel, ‘bed-side’ point-of-care nanopore sequencing technology for real-time rapid identification of microbial keratitis.
An ex-vivo porcine model (n=60) of microbial keratitis was developed to compare DNA recovery rates of 11 different collection methods: 25-gauge needle, 4mm PTFE membrane, Isohelix SK2, Surgi®Eyespear, Cotton, Rayon, Dryswab™, Hydraflock, Albumin-coated, Purflock, and Polyester swabs.DNA was extracted, 16S rRNA genes amplified, sequenced on GridION and analysed using Epi2Me workflow. Differences in DNA recovery rates were compared using Kruskal-wallis test. Minimum abundance of 3% cut off was used to generate NCBI Taxonomy Tree.The collection method with the highest DNA recovery was used to collect corneal samples from patients presenting with suspected microbial keratitis. For each patient, swabs were taken from each eye together with a negative control ‘air swab’ of the clinical room at the time point of patient sampling. Sequence data were compared with corresponding hospital corneal scrapes microbiology culture results.
DNA concentration recovery rates were significantly different between the collection methods (p<0.001). Surgi®Eyespear cellulose biomaterial (Total read count: 139,604 reads; median DNA concentration:82.2ng/ul per swab; IQR:40.7-221.1ng/ul), Cotton (151,111 reads; 30; 10.26-61.6ng/ul), and Polyester (20,154 reads; 2.37; 1.62-56.3ng/ul) swabs provided the highest DNA recovery rates, whilst needle and PTFE membrane gave the lowest DNA yield.Surgi®Eyespear was used to collect corneal swabs from two consecutive patients with suspected microbial keratitis. A total of 1523950 reads were generated, with average accuracy of 86%. Reads corresponding to pathogens were identified within the first 2 hours from initiation of sequencing. Patient 1 had a majority of reads mapping to Serratia marcescens which was in concordance with culture results from corneal scrapes. Patient 2 did not have any dominant bacterial taxa on sequencing that met significance, in concordance with negative culture results.
We demonstrate a novel, rapid and portable workflow for real-time identification of bacterial microbial keratitis using nanopore sequencing.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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