New insights into culture negative endophthalmitis by unbiased next generation sequencing

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.

Figure 1

Observed Rarefaction curves calculated using the OTU matrix obtained from the Illumina HiSeq raw data by using QIIME are shown for all the analysed samples and sequencing runs. OTUs diversity for each sample obtained (Left) using V3–V4 primers (Right) ITS2 primers.

Figure 2

Stacked box-plot displaying the relative abundances of the bacterial taxa (≥3%) and species richness obtained by NGS on vitreous samples of (a) Culture positive endophthalmitis (b) Culture Negative endophthalmitis (c)Stacked box-plot displaying the relative abundances of the fungal taxa (≥3%) and species richness obtained by NGS on vitreous samples of both Culture positive (#2) and Culture Negative samples (#8,12,6).

Figure 3

Heat map of Genera Level Bacterial Abundances in samples from patients with presumed infectious both culture positive and culture negative endophthalmitis. The color bar on the right side indicates the average relative abundances of these genera in each patient.