Clinical SciencePythium insidiosum Keratitis Clinical Profile and Role of DNA Sequencing and Zoospore Formation in DiagnosisSharma, Savitri MD; Balne, Praveen K. MSc; Motukupally, Swapna R. MD; Das, Sujata MS, FRCS(Glasg); Garg, Prashant MS; Sahu, Srikant K. MS; Arunasri, Kotakonda MSc; Manjulatha, Kodiganti MSc; Mishra, Dilip K. MD; Shivaji, Sisinthy PhDAuthor Information *Jhaveri Microbiology Centre, Hyderabad Eye Research Foundation, L. V. Prasad Eye Institute, Hyderabad, India; †Cornea and Anterior Segment Services, Hyderabad Eye Research Foundation, L. V. Prasad Eye Institute, Bhubaneswar, India; ‡Cornea and Anterior Segment Services, Hyderabad Eye Research Foundation, L. V. Prasad Eye Institute, Hyderabad, India; §Indian Institute of Chemical Technology, Hyderabad, India; and ¶Ophthalmic Pathology Service, Hyderabad Eye Research Foundation, L. V. Prasad Eye Institute, Hyderabad, India. Reprints: Savitri Sharma, MD, Jhaveri Microbiology Centre, Brien Holden Eye Research Centre, Hyderabad Eye Research Foundation, L. V. Prasad Eye Institute, Rd No. 2, Banjara Hills, Hyderabad 500034, India (e-mail: [email protected]). Hyderabad Eye Research Foundation, Hyderabad, India. Council of Scientific and Industrial Research, Government of India, New Delhi, India [grant 27(0191)/09/EMR-ΙΙ]. P.K. Balne, Jhaveri Microbiology Centre, was supported by a senior research fellowship from the Council of Scientific and Industrial Research (CSIR), Government of India, New Delhi, India. The authors have no conflicts of interest to disclose. Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Web site (www.corneajrnl.com). Received June 13, 2014 Received in revised form November 11, 2014 Accepted November 12, 2014 Cornea: April 2015 - Volume 34 - Issue 4 - p 438-442 doi: 10.1097/ICO.0000000000000349 Buy SDC Metrics Abstract 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. Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.