Pythium insidiosum keratitis: clinical profile and role of DNA sequencing and zoospore formation in diagnosis.

PURPOSE: To report the molecular and microbiological diagnosis and clinical profile of 13 patients with Pythium insidiosum keratitis. METHODS: Phase 1 of the study consisted of DNA sequencing of the ITS region of the rDNA of 162 stocked morphologically unidentified nonconsecutive fungal isolates from corneal scraping of patients with keratitis (2010-2012). Blast and phylogenetic analyses of the sequences showed 9 to be P. insidiosum. A retrospective review of archived photographs of colony and direct microscopy of corneal scrapings and clinical records of the cases were performed. Phase 2 began in 2014, in which a simple method of zoospore formation was used for fungal colonies resembling those of P. insidiosum followed by DNA sequencing. RESULTS: The prevalence of P. insidiosum among unidentified fungal isolates from keratitis was 9/162 (5.5%) in phase 1. In phase 2, 4/102 cases (3.9%) of fungal keratitis were identified as P. insidiosum (January-February, 2014). Phylogenetic analysis of all 13 fungal isolates confirmed the identification of P. insidiosum. Corneal infiltrates exhibited hyphate edges, tentacle-like extensions, and dot-like infiltrates surrounding the main infiltrate. Response to topical 5% natamycin eye drops with or without oral antifungals was poor (penetrating keratoplasty: 9 and evisceration: 2) with a mean follow-up period of 82 days. CONCLUSIONS: P. insidiosum keratitis needs to be considered in the differential diagnosis of severe fungal keratitis. It can be identified using the zoospore formation method and confirmed by ITS DNA sequencing. Lack of response to currently used antifungal drugs calls for evaluation of newer drugs for medical therapy and consideration for early penetrating keratoplasty.