Abstract
The proof-of-concept, study to investigate the presence of microorganisms in presumed infectious endophthalmitis using Next generation sequencing (NGS) was carried out in vitreous biopsies from 34 patients with endophthalmitis, and thirty patients undergoing surgery for non-infectious retinal disorders as controls. Following DNA extraction using the Qiagen mini kit and PCR amplification of the V3–V4 regions of the bacterial 16S rRNA and ITS 2 region of fungus, they samples were sequenced on an Illumina HiSeq 2500 Machine. Paired reads were curated, taxonomically labeled, and filtered. Culture based diagnosis was achieved in 15/34 (44%) patients while NGS diagnosed the presence of microbes in 30/34 (88%) patients (bacteria in 26/30, fungi in 2/30, mixed infections in 2/30 cases). All 30 controls were negative for bacteria or fungus by NGS. There was good agreement between culture and NGS for culture-positive cases. Among culture negative cases, DNA of common culturable bacteria were identified like Streptococcus sp., Staphylococcus sp., Pseudomonas sp., Gemella sp., Haemophilus sp., Acinetobacter sp. The specificity of NGS with culture and clinical diagnosis was found to be 20% and 100% respectively and sensitivity of NGS with culture and clinical diagnosis was found to be 87.5% and 88% respectively. NGS appears to be promising diagnostic platform for the diagnosis of infectious culture negative endophthalmitis.
Highlights
Endophthalmitis, is a potentially sight-threatening condition that varies geographically in incidence and in cause, following surgical procedures, trauma or endogenous dissemination[1]
The most common risk factor linked with presumed infectious endophthalmitis was trauma in 21 (61.7%) patients followed by cataract surgery in 7 patients (20.5%), endogenous source in 2 cases, non resolving fungal keratitis in 2 cases and in remaining 2 cases it was unknown
We report the outcome of a proof-of-concept study that uses generation sequencing on vitreous fluid for the identification of bacteria and fungi in patients with presumed infectious endophthalmitis as well as to understand the genesis and surreptitiousness of culture-negative endophthalmitis
Summary
Endophthalmitis, is a potentially sight-threatening condition that varies geographically in incidence and in cause, following surgical procedures, trauma or endogenous dissemination[1]. Next-generation sequencing (NGS) is assumption-free, meaning that it does not target just one specific species but can detect all the different fungi/bacteria present in a clinical sample in one single assay. This technique promises improved detection of traditional organisms but can has the potential to identify newer organisms not previously associated with endophthalmitis. Accurate diagnosis of endophthalmitis using generation sequencing may makes it feasible to determine a better treatment startegy in these cases, but it may improve outcome in culture negative cases in which delayed diagnosis www.nature.com/scientificreports/. Has likely contributed to historically poor outcomes and may become the new standard in the management of intraocular infections
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