Abstract
Purpose :
Advances in next-generation sequencing (NGS) have provided the opportunity for developing a comprehensive method to identify infectious agents. In this study, we evaluated the diagnostic utility of target-specific primers for PCR-mediated amplification with the NGS technology, from vitreous samples of patients diagnosed with acute post-operative endophthalmitis, especially for culture negative cases.
Methods :
Undiluted vitreous fluid was collected from patients with clinical diagnosis of post-operative (cataract surgery) endophthalmitis from April 2019 - April 2023 and processed by traditional microbiology culture according to institute protocol. A portion of the samples was used for DNA extraction using a Qiagen mini kit followed by amplification of V3–V4 regions of the 16s rRNA gene of the bacterial and ITS 1 and ITS 4 regions of the fungal genome by PCR. Half of the amplicons were processed for bidirectional Sanger sequencing using the Applied Biosystems 3500xL; the remaining was processed for deep sequencing on the Illumina HiSeq 2500 machine, and the data was analysed using QIIME and R software.
Results :
In this cohort of 300 patients, 69 vitreous samples were culture-positive (23%) and included 62 bacteria-positive and 7 fungus-positive. Fifty-seven culture-negative samples were PCR-positive for bacterial or fungal DNA by PCR. In these culture-negative PCR-positive vitreous samples, Sanger sequencing identified microorganisms in 38 instances (66.6% of culture-negative vitreous) and included 29 bacteria and 9 fungi. The NGS identified microorganisms in all 57 culture-negative PCR-positive vitreous samples; it included 45 bacteria (predominantly Staphylococcus spp, n=14) and 12 fungi (predominantly Aspergillus spp, n=3). Additionally, NGS detected polymicrobial infections in 42.1% (24/57) culture-negative samples and 20.3% (14/69) culture-positive samples, which could not be detected using Sanger sequencing.
Conclusions :
NGS is an evolving molecular tool for identifying pathogens in postoperative endophthalmitis. Deep sequencing can catalogue bacterial species in mixed specimens from which conventional clinical methods cannot obtain usable data, and it is advantageous in culture-negative cases and polymicrobial infection in postoperative endophthalmitis.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.