Microbiological Culture Versus 16S/18S Ribosomal RNA PCR-Sanger Sequencing for Infectious Keratitis: A Three-Arm, Diagnostic Cross-Sectional Study

Abstract

Purpose: To compare the diagnostic performance of microbiological culture and 16S/18S polymerase chain reaction (PCR) for infectious keratitis (IK). Methods: This was a monocentric, three-arm, diagnostic comparative study. We included 81 patients (86 episodes of IK) who presented with presumed bacterial/fungal keratitis to the Queens Medical Centre, Nottingham, UK, between June 2021 and September 2022. All patients underwent simultaneous microbiological culture (either direct culture, indirect culture, or both) and 16S (pan-bacterial)/18S (pan-fungal) ribosomal RNA (rRNA) PCR-Sanger sequencing. Main outcome measures included diagnostic yield, performance, and inter-test agreement. Results: All organisms identified were of bacterial origin. Diagnostic yields were similar among direct culture (52.3%), indirect culture (50.8%), and PCR (43.1%; p=0.13, Cochran Q test). The addition of PCR enabled a positive diagnostic yield in 3 (9.7%) direct culture-negative cases. Based on composite reference standard, direct culture had the highest sensitivity (87.5%; 95% CI, 72.4-95.3%), followed by indirect culture (85.4%; 95% CI, 71.6-93.5%) and PCR (73.5%; 95% CI, 59.0-84.6%), with 100% specificity noted in all three tests. Pairwise comparisons showed substantial agreement among the three tests (percent agreement=81.8-86.2%, Cohen k=0.67-0.72). Clinico-microbiological correlation demonstrated higher culture-PCR concordance in cases with worse presenting visual acuity and greater severity of infection. Conclusion: This study highlights a similar diagnostic performance of direct culture, indirect culture and 16S rRNA PCR for bacterial keratitis, with substantial inter-test concordance. PCR serves as a useful diagnostic adjuvant to culture, particularly in culture-negative cases or those with lesser disease severity (where culture-PCR concordance is lower).