New insights into culture negative endophthalmitis by unbiased next generation sequencing

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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.

Introduction

Endophthalmitis, is a potentially sight-threatening condition that varies geographically in incidence and in cause, following surgical procedures, trauma or endogenous dissemination1. The ability to identify the causative organism has huge implications in treatment and clinical management of the patients. Extensive variation in culture positivity from 38–44% in clinically diagnosed endophthalmitis cases is known and has been reported in Indian subjects2,3,4. The aetiologic agent is therefore unknown in a majority of patients, when routine aerobic bacterial cultures are negative. Culture-negativity could be attributed to fastidious nature of the inciting organism that may be difficult to grow in culture or may be unculturable. Inspite of the limitation of low yield and inability of certain pathogens to grow on routine media, microbiological culture still remains the current gold standard for the diagnosis of most intraocular infections. The use of molecular tools like PCR has improved the yield of detection in addition to reducing the time to make a confirmatory diagnosis5. However the number of fungal and/or bacterial pathogens that can be simultaneously detected is limited, due to differences in amplification efficiencies of different primer sets and the limited number of fluorescent labels because of which multiplexing different PCRs assays becomes technically challenging.

In comparison, 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 not only improved detection of traditional organisms but can also has the potential to identify newer organisms not previously associated with endophthalmitis. Recent reports of the presence of Torque Teno Virus in the vitreous of patients diagnosed with endophthalmitis has substantiated this claim6. We would like to test the feasibility of the application of next generation DNA sequencing to vitreous biopsies from patients with endophthalmitis and compare the results with traditional culture techniques. Accurate diagnosis of endophthalmitis using next generation sequencing may not only makes it feasible to determine a better treatment startegy in these cases, but it may also improve outcome in culture negative cases in which delayed diagnosis has likely contributed to historically poor outcomes and may become the new standard in the management of intraocular infections.

Results

A total of 34 presumed infectious endophthalmitis – aqueous/vitreous biopsies and 30 vitreous control samples were included in the study. There were 22 males and 12 females in the test group and the mean age was 35 ± 25.6 years. Clinical and demographic details of patients are given in Table 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. Initially patients were treated with injection of intravitreal antibiotics (vancomycin 1 mg/0.1 ml and ceftazidime 2.25 mg/0.1 ml) with or without intravitreal dexamethasone (400 μg/0.1 ml) in addition to pars plana vitrectomy (PPV) in 25 (73.5%) patients and/or lensectomy and vitrectomy along with intravitreal antibiotics in 9 (26.4%) of the 34 patients. In 3 patients there was a clinical suspicion of fungal infection and additional intravitreal amphotericin B (5 µg/0.1 ml) was given while one patient (#25) was given intravitreal and intracameral linezolid as it was a suspected case of Pythium keratits. Two patients had an intraocular foreign body removal (IOFB-R) while one required intraocular lens (IOL) to be explanted. The medical treatment also included intensive mainly ciprofloxacin (0.3% 1 hourly) and prednisolone acetate (1% one hourly) along with oral ciprofloxacin 750 mg (2/day for 7–10 days). The 5 year-old child (#29) was given cephalexin syrup (125 mg twice daily × 6 days) and intravitreal gentamicin. None of the patients were clinically immunocompromised. However, we had two patients with endogenous endophthalmitis who were children aged one and six years and it is possible that prior to reporting to our hospital with endogenous endophthalmitis the children were ill, the details of whch were not available to us. Post-treatment 7/34 (20.5%) patients had a visual outcome of 20/200 or better.

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