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.

Background: Pythium insidiosum is a parafungus that causes keratitis resembling fungal keratitis. This study compares outcome in a large cohort of patients with P insidiosum keratitis treated with antifungal drugs, to a pilot group treated with antibacterial antibiotics.Methods: Between January 2014 and December 2016, 114 patients with culture positive P insidiosum keratitis were included in the study. A subset of culture isolates was tested in vitro for response to nine antibacterial antibiotics by disc diffusion and E test. Patients were treated with topical natamycin in 2014, 2015 and up until mid 2016. Thereafter, the patients received a combination of topical linezolid and topical and oral azithromycin. Therapeutic penetrating keratoplasty (TPK) was done for patients not responding to medical therapy.Results: In vitro disc diffusion assay showed linezolid to be most effective. The rate of TPK was significantly higher in 2015 compared with 2016 (43/45, 95.6% vs 22/32, 68.8%; p=0.002). Eighteen patients were treated with antibacterial and 14 were treated with antifungal antibiotic in 2016. One patient was lost to follow-up in each group. The rate of TPK was higher and proportion of healed ulcers was lower (p=0.21, Fisher’s exact test) in the group on antifungal therapy (TPK—11/13, 84.6%; Healed—2/13, 15.3%) compared with the group on antibacterial therapy (TPK—11/17, 64.7%; Healed—6/17, 35.2%).Conclusions: We report favourable but not statistically significant response of P insidiosum keratitis to antibacterial agents in a pilot series of patients. Further evaluation of this strategy in larger number of patients is recommended.