Advances in the laboratory diagnosis of non-viral microbial keratitis

Infectious keratitis remains a leading cause of corneal blindness and visual impairment worldwide, with bacterial, fungal, amoebic and viral pathogens presenting major diagnostic and therapeutic challenges. Early and precise pathogen identification is essential to initiate early treatment and prevent irreversible ocular damage. Direct visualisation of pathogens by microscopy of corneal scrapings has been one of the most rewarding and time-tested methods; however, ocular microbiology laboratories seem to be losing the expertise for the same. Culture methods also seem to be losing favour for their long turnaround time and low sensitivity. These have paved the way for newer, rapid and sensitive molecular techniques to make a foray into the realm of diagnosis of non-viral keratitis. This review critically evaluates recent advances in molecular diagnostics for infectious keratitis, focusing on DNA/RNA-based sequencing, real-time PCR (qPCR) and mass spectrometry (MS) and examines their clinical relevance, limitations and potential for point-of-care application.An extensive literature analysis was conducted, integrating findings from peer-reviewed studies published between 2000 and 2025. Emerging diagnostic platforms were compared with conventional microbiological methods in terms of diagnostic accuracy, turnaround time and clinical applicability across paediatric and adult cohorts. Traditional culture-based approaches exhibit limited sensitivity (30%-60%) and slow pathogen detection. In contrast, multiplex and quantitative PCR enable rapid, sensitive and specific pathogen identification, including in culture-negative cases. Next-generation sequencing offers broad-spectrum, culture-independent profiling of bacteria, fungi and protozoa, enhancing diagnostic yield and understanding of pathogen diversity. MS has transformed microbial and host proteomic/metabolomic analyses, identifying candidate biomarkers for disease severity and prognosis. Recent innovations such as portable qPCR devices, clustered regularly interspaced short palindromic repeats-based diagnostics and artificial intelligence-assisted data interpretation extend molecular testing to near-patient settings. The integration of these technologies into routine clinical workflows promises earlier intervention, personalised therapy and improved visual outcomes, particularly in resource-limited environments.