16S/18S metabarcoding versus culture for aetiological assessment in infectious keratitis: A prospective paired-sample study.

Abstract

Purpose: Infectious keratitis (IK) is a vision-threatening condition with diverse microbial aetiologies, yet conventional culture-based diagnostics often have limited sensitivity. We investigated whether 16S/18S rRNA amplicon-based next-generation sequencing (NGS) can enhance aetiological assessment when used alongside standard microbiological methods.

Methods: In this prospective paired-sample study, 57 patients with IK underwent corneal sampling with conventional culture, PCR (HSV/VZV and Acanthamoeba) and 16S/18S metabarcoding. Microbiological findings were interpreted in a structured workflow incorporating clinical correlation and comparison with samples from the contralateral healthy eye.

Results: Excluding entirely viral cases, NGS did not significantly increase the overall diagnostic outcome compared with culture and PCR alone (n = 53, 42% vs. 51%, p = 0.27) but improved the combined diagnostic yield (n = 53) to 58%. NGS identified clinically relevant pathogens in 39% of cases, including four cases not detected by culture, whereas culture identified nine cases missed by NGS. Concordance between methods was observed in 55% of positive cases. NGS on samples from the contralateral eye aided interpretation of NGS data in over 25% of cases by distinguishing commensal background from likely pathogens. Although microbiological findings led to treatment modification in 16% of cases, these were all in cases diagnosed by conventional methods.

Conclusion: Based on our findings, 16S/18S metabarcoding may be useful as a targeted supplementary test in atypical, refractory or culture-negative IK cases where conventional sampling is compromised. Findings should be interpreted alongside conventional microbiology, clinical findings and preferably contralateral-eye sampling.