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

Abstract

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.

Keywords: broad-range PCR; ocular infections; real-time PCR.

Figure 1

Agarose gel electrophoresis of conventional PCR products. Lane L represents the size ladder; lane C represents the positive control; lanes S1 and S2 represent ocular specimens 1 and 2, respectively.

Figure 2

Comparison of ΔCT values of the ocular samples stored in different solutions. The thioglycolate-stored specimens had a significantly lower ΔCT value compared with the specimens stored in distilled water (p = 0.01105). Abbreviations: DW, distilled water; Thio, thioglycolate broth.

Figure 3

ROC curve analysis of ΔCT values. (A) Aseptic specimens and (B) septic specimens, compared with the culture results of occasional or more than occasional colonies. Abbreviations: Sens, sensitivity; Spec, specificity; PPV, positive predictive value; NPV, negative predictive value; AUC, area under the curve.

Figure 4

Concordance of real-time PCR results, shown by the time interval between sample collection and nucleic acid extraction.