Enhanced Detection of Bacterial Ocular Pathogens: A Comparative Study of Broad-Range Real-Time PCR and Conventional...

Background: Ocular infections can cause severe complications, including blindness, and distinguishing bacterial from fungal keratitis based on clinical features alone is difficult. This study compared broad-range conventional PCR and real-time PCR methods targeting the 16S rRNA gene with traditional culture for diagnosing bacterial ocular infections. Methods: We analyzed 160 ocular specimens from 111 patients, categorizing them as septic or aseptic. The results of both conventional PCR and real-time PCR methods targeting the 16S rRNA gene were compared with traditional culture outcomes. Results: Real-time PCR demonstrated higher sensitivity than conventional PCR, and receiver operating characteristic analysis determined optimal ΔCT cutoff values of -2.13 and -4.09 for septic and aseptic specimens, respectively. Delays in specimen processing significantly affected real-time PCR accuracy. The 16S rRNA meta-taxonomic analysis using nanopore sequencing only validated the PCR results when the DNA concentration was sufficient. Conclusions: Broad-range real-time PCR proved to be a valuable diagnostic tool, particularly in aseptic specimens, with greater sensitivity and specificity than conventional PCR. The established ΔCT cutoff values improved diagnostic accuracy and showed that standardized specimen collection and processing are crucial for maximizing PCR efficacy.