Innovations in the diagnosis of Pythium insidiosum keratitis: molecular advances and the path to point-of-care...

Pythium insidiosum keratitis is an aggressive, vision-threatening corneal infection that closely mimics fungal keratitis clinically and on smear microscopy, frequently delaying targeted therapy and appropriate surgical intervention. Rapid and reliable molecular diagnosis is therefore critical, particularly given the small-volume, low-biomass nature of corneal specimens encountered in routine ophthalmic practice worldwide, especially in endemic regions with limited diagnostic infrastructure.

This review summarizes advances in molecular diagnostics for Pythium insidiosum keratitis, including conventional and nested PCR, duplex and multiplex PCR, real-time PCR, high-resolution melt analysis, loop-mediated isothermal amplification (LAMP), colorimetric LAMP, metagenomic sequencing, and CRISPR-based platforms. Key diagnostic targets such as ITS, 18S rRNA, COX2, and PinsEXO1 are evaluated for analytical sensitivity, specificity, turnaround time, contamination control, and applicability in endemic, resource-limited settings and peripheral ophthalmic centers with limited infrastructure and limited availability of trained personnel.

Future progress will depend on integrating robust molecular targets with closed-tube, contamination-resistant amplification platforms within tiered diagnostic systems. Colorimetric LAMP offers immediate translational potential, while PinsEXO1-based assays enhance specificity. CRISPR-based detection and microfluidic integration remain promising but require rigorous validation in ocular specimens before routine clinical adoption in diverse real-world settings globally, across varied healthcare systems and populations.