Time to improve laboratory protocols in microbial keratitis: broad-range Internal Transcribed Spacer sequencing in refractory culture-negative keratitis.

A woman in her 60s who was a soft contact lens wearer presented with rapidly progressive microbial keratitis unresponsive to intensive empirical therapy. Despite extensive investigations - including conventional cultures, impression cytology, in vivo imaging, and repeated targeted PCR from corneal and aqueous samples - the aetiology remained unclear, with all tests persistently negative. Progressive inflammation, enlarging hypopyon, stromal melt and rising intraocular pressure necessitated anterior chamber (AC) washout followed by therapeutic penetrating keratoplasty with extracapsular cataract extraction. Histopathology and PCR from the excised button were again non-diagnostic. When new epithelial breakdown and an AC mass raised concern for ongoing recrudescence of the infection, a loose corneal suture was removed and analysed: broad-range ITS2 rRNA sequencing detected fungal DNA consistent with Fusarium, providing the first microbiological clue after weeks of negative investigations although not corroborated by other tests and guiding targeted antifungal therapy. This case underscores the limitations of standard microbiology and targeted PCR in deep, pre-treated keratitis and highlights the value of broad-range Internal Transcribed Spacer sequencing as a critical complementary diagnostic tool when conventional methods repeatedly fail.