Identification of Bacterial and Fungal Pathogens by rDNA Gene Barcoding in Vitreous Fluids of Endophthalmitis Patients

Abstract

ABSTRACT Purpose: To identify the bacterial and fungal pathogens in ocular samples of clinically suspected endophthalmitis patients by conventional culture methods and 16S and 28S rDNA gene sequencing respectively. Methods: A total of 88 patients with clinically suspected endophthalmitis were included in this study. Under sterile operating conditions, a vitreous fluid (0.1–0.2 ml) was obtained by pars plana vitrectomy procedure. The samples were processed for conventional microbiology methods and PCR. PCR targeting 16S rDNA gene for bacteria and 28S rDNA gene for fungus were performed individually using the MightyAmp DNA Polymerase Ver. 2 (TaKaRa China) kit. The PCR amplified samples were sequenced and aligned using CLUSTAL-W tool. The phylogenetic tree was constructed by Neighborhood joining along with the reference sequences downloaded from NCBI database using MEGA X software. Results: 67 Post-operative, 12 Endogenous and 9 traumatic endophthalmitis patients were included as study subjects. By the direct culturing bacterial growth was observed in 17 samples and fungal growth in three samples. PCR was positive for all the culture positive samples, in addition 14 were positive in culture negative samples. The predominant species identified in gram-positive bacteria were Staphylococcus spp., and Pseudomonas spp. in the gram-negative group. Both PCR and culture identified only three samples positive for fungal pathogens which were identified as Aspergillus fumigatus, Candida albicans, and Exerohilum rostratum. Conclusions: PCR based molecular diagnosis is more sensitive than the conventional gold standard culture methods in endophthalmitis. Bacterial pathogens were found to be the predominant in causing endophthalmitis than fungal pathogens.