ena-STUDY-UNIVERSITY OF BIRMINGHAM-24-07-2019-08:21:18:306-1865
Rapid Diagnosis of Microbial Keratitis using Nanopore Sequencing: a proof-of-concept study
PurposeMicrobial keratitis is a leading cause of blindness worldwide. Prognosis is dependent upon rapid diagnosis of causative pathogen and early treatment initiation. We aim to investigate and optimise the use of a novel, 'bed­side' point­of­care nanopore sequencing technology for real­time rapid identification of microbial keratitis.MethodsAn ex­vivo porcine model (n=60) of microbial keratitis was developed to compare DNA recovery rates of 11 different collection methods: 21­gauge needle, PTFE membrane, Isohelix SK2, Sugi®Eyespear, Cotton, Rayon, Dryswab, Hydraflock, Albumin­coated, Purflock, and Polyester swabs. 16S bacterial to host DNA recovery ratio was determined using qPCR. 16S rRNA genes were amplified, sequenced on GridION and analysed using Epi2Me workflow. The collection method with the highest DNA recovery was used to collect corneal samples from patients presenting with suspected microbial keratitis. Sequence data were compared with corresponding hospital corneal scrape microbiology culture results.ResultsDNA concentration recovery rates were significantly different between the collection methods (p<0.001). Sugi®Eyespear, Isohelix SK-2 and Cotton swabs provided the highest DNA recovery, whilst needle and PTFE membrane gave the lowest DNA yield.Sugi®Eyespear was used to collect corneal swabs from two consecutive patients with suspected microbial keratitis. Patient 1 had a majority of reads mapping to Serratia marcescens, which was in concordance with culture results from corneal scrapes. Patient 2, with negative culture results, showed a predominance of Bacillus genera on sequencing, in concordance with MiSeq sequencing results.ConclusionsWe have optimised collection methods and demonstrate a novel, rapid and portable workflow for real-time identification of bacterial microbial keratitis using nanopore sequencing.
PurposeMicrobial keratitis is a leading cause of blindness worldwide. Prognosis is dependent upon rapid diagnosis of causative pathogen and early treatment initiation. We aim to investigate and optimise the use of a novel, 'bed­side' point­of­care nanopore sequencing technology for real­time rapid identification of microbial keratitis.MethodsAn ex­vivo porcine model (n=60) of microbial keratitis was developed to compare DNA recovery rates of 11 different collection methods: 21­gauge needle, PTFE membrane, Isohelix SK2, Sugi®Eyespear, Cotton, Rayon, Dryswab, Hydraflock, Albumin­coated, Purflock, and Polyester swabs. 16S bacterial to host DNA recovery ratio was determined using qPCR. 16S rRNA genes were amplified, sequenced on GridION and analysed using Epi2Me workflow. The collection method with the highest DNA recovery was used to collect corneal samples from patients presenting with suspected microbial keratitis. Sequence data were compared with corresponding hospital corneal scrape microbiology culture results.ResultsDNA concentration recovery rates were significantly different between the collection methods (p<0.001). Sugi®Eyespear, Isohelix SK-2 and Cotton swabs provided the highest DNA recovery, whilst needle and PTFE membrane gave the lowest DNA yield.Sugi®Eyespear was used to collect corneal swabs from two consecutive patients with suspected microbial keratitis. Patient 1 had a majority of reads mapping to Serratia marcescens, which was in concordance with culture results from corneal scrapes. Patient 2, with negative culture results, showed a predominance of Bacillus genera on sequencing, in concordance with MiSeq sequencing results.ConclusionsWe have optimised collection methods and demonstrate a novel, rapid and portable workflow for real-time identification of bacterial microbial keratitis using nanopore sequencing.