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.

Third-Generation Sequencing in the Clinical Laboratory: Exploring the Advantages and Challenges of Nanopore Sequencing

“…The use of NGS for infectious disease diagnostics is being increasingly explored and adopted for select applications in the clinical laboratory (4,(7)(8)(9)(10)(11)(12). Several approaches to pathogen detection with NGS can be undertaken (Table 2).…”

Targeted High-Throughput Sequencing Identifies Predominantly Fungal Pathogens in Patients with Clinically Infectious, Culture-Negative Endophthalmitis in South India

“…To cross the divide between the conventional microbiological methods and whole-genome sequencing, a culture free platform using targeted HTS would be an excellent, less complicated approach, with a lower cost to detect pathogens for implementation in diagnostic laboratories. We have previously shown that NGS is a good tool for microbial research in endophthalmitis [9], but the next big challenges will be the standardization and validation of procedures and bioinformatics pipeline that address the specific challenges for clinical standards, especially in culture-negative cases. HTS is more likely to become a future diagnostic tool in routine ocular microbiological laboratories.…”

Introductory Chapter: Current and Future Trends in the Diagnosis and Management of Uveitis

“…More recently, the use of next-generation sequencing (NGS) has proven to be a promising diagnostic strategy for multiple detections of common and rare microorganisms, including virus associated with infectious uveitis and endophthalmitis present in single vitreous samples. An important contribution of NGS so far is related to the improvement of pathogen detection in cases of negative culture endophthalmitis [8].…”