BASIC INVESTIGATION Pythium Insidiosum Keratitis: Histopathology and Rapid Novel Diagnostic Staining Technique Ruchi Mittal, MD, DNB,*† Shipra K. Jena, MSc,* Alisha Desai, MS,† and Sunil Agarwal, MD‡ Key Words: Pythiuim insidiosum keratitis, fungal keratitis, oomy- Purpose: To elucidate the histopathology of Pythium insidiosum cete, histopathology, iodine–potassium iodide–sulfuric acid stain keratitis and to describe a novel, simple, and rapid staining technique for identification of oomycete Pythium insidiosum and to differen- (Cornea 2017;36:1124–1132) tiate it from fungi. Methods: This is a laboratory investigation study of 38 non- ythium insidiosum, a pathogenic oomycete is the only consecutive cases (37 ocular samples and 1 colonic biopsy); 14 Preported agent to cause pythiosis in mammals, primarily microbiologically diagnosed as Pythium insidiosum keratitis and 24 causing disease in horses, dogs, and humans. Human as fungal keratitis. Review of clinical, demographic details, micro- pythiosis manifests itself as cutaneous, corneal, orbital, fi biological results, and identi cation of cases that necessitated vascular, gastrointestinal, and systemic forms of infection, evisceration was performed. Reevaluation of histopathology slides which can be devastating or life threatening.1–13 Pythium – was done using stains such as hematoxylin eosin, Gomori methe- infection is associated with high morbidity and mortality, and – – namine silver (GMS), periodic acid Schiff (PAS), potassium iodide its timely diagnosis and treatment is a chief cause for concern. sulfuric acid (IKI-H2SO4). Morphology, degree, and nature of Ocular pythiosis, commonly presents with corneal fl in ammation and load, distribution, and staining results of Pythium involvement and affects healthy individuals.5 The major insidiosum and its comparison with fungi were studied. challenge in management lies in the fact that it is misdiagnosed Results: and treated as a fungal infection, not only because of Delay in zoospore formation, failure of growth, and delay fi in identification of Pythium were the main cause of evisceration. insuf cient awareness among medical and laboratory specialists Corneal pythiosis showed epithelial ulceration, stromal destruction, and its morphological resemblance to fungi but also because of fl the lack of simple, quick, cost-effective, highly sensitive, and and varying in ammation; load and distribution of Pythium were fi inversely proportional to inflammation. The filaments were com- speci c diagnostic methods. Currently, there are no standard monly wide, with admixed narrower structures and uncommonly treatment protocols for pythiosis, and radical surgeries are involved Descemet membrane. The oomycete was not discretely advocated to completely excise the infected tissue to control the 8,14 discerned with PAS stain and stained distinctly with GMS stain and disease. Although oomycete Pythium insidiosum resembles IKI-H SO stain (100% sensitive). In comparison, fungal organisms fungi, under light microscopy it differs in its basic structural 2 4 composition and thus does not respond to antifungal medical stained well with PAS and GMS stain, but not with IKI-H2SO4 stain (100% specific). therapy. It contains cellulose in its cell wall, unlike fungi, which are chitinous and lack cellulose. The cell membrane of Pythium Conclusions: Pythium insidiosum keratitis is perhaps not more lacks ergosterol, unlike a fungal organism.15,16 devastating than fungal keratitis but late diagnosis, misdiagnosis, and It is imperative for all laboratory professionals to be treatment as fungal infection are major heralds. Early diagnosis may aware of the morphology and distinctive identification markedly improve the patient outcome. IKI-H2SO4 is a cost- features of this pathogenic oomycete. Zoospore formation, effective, simple, sensitive, and specific stain for the diagnosis of immunohistochemical detection, noncommercial serological oomycete Pythium. tests, and gene sequencing techniques have been studied for its diagnosis and differentiation from fungal organisms.5,6,17–23 Although zoospore formation is recommended as an important Received for publication February 6, 2017; revision received April 4, 2017; fi 5,17 accepted April 9, 2017. Published online ahead of print June 2, 2017. diagnostic tool for identi cation of Pythium, it is known to From the *Dalmia Ophthalmic Pathology Services, L. V. Prasad Eye Institute, be fraught with limitations. In addition to a delay period of 3 Bhubaneswar, India; †Tej Kohli Cornea Institute, L. V. Prasad Eye to 7 days, absence of growth in media in more than 30% of Institute, Bhubaneswar, India; and ‡Department of Pathology, Kalinga cases5 can limit its identification by zoospore formation or Hospital Limited, Bhubaneswar, India. molecular diagnostic tools. In addition, zoospore formation Supported by Hyderabad Eye Research Foundation and Tej Kohli Cornea Institute. cannot be demonstrated if the biopsy material is sent to the The authors have no conflicts of interest to disclose. histopathology laboratory alone. S. K. Jena has equal contribution as first author. In such a scenario, it is imperative that professionals are Reprints: Ruchi Mittal, MD, DNB, Dalmia Ophthalmic Pathology Services adequately trained and equipped to accurately identify and Tej Kohli Cornea Institute, L. V. Prasad Eye Institute, SMTC Campus, Patia, Bhubaneswar 751024, Orissa, India (e-mail: dr.rmittal@ Pythium from all laboratory samples, including surgically gmail.com). resected formalin-fixed paraffin-embedded (FFPE) tissues; Copyright © 2017 Wolters Kluwer Health, Inc. All rights reserved. hence, we performed a study to identify oomycete Pythium 1124 | www.corneajrnl.com Cornea Volume 36, Number 9, September 2017 Copyright Ó 2017 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited. Cornea Volume 36, Number 9, September 2017 Pythium Insidiosum Keratitis insidiosum using simple staining techniques and also to study (PA). All the slides were treated in PA (0.5% and 1.0%) in the morphological features of Pythium insidiosum keratitis. various batches for 0.5, 2, 3, and 5 minutes. Schiff reagent was used without any modifications for 10 minutes. The difference MATERIALS AND METHODS in the intensity of staining was noted as negative, 1+ to 4+ with different concentrations and durations of staining. Inability to The study was conducted at L. V. Prasad Eye Institute, visualize the filamentous structures was recorded as negative. India. Informed consent was obtained from all patients, and the When the structures appeared pink, similar to stromal staining, study was approved by the institutional review board, reference it was called as 1+. Intensity of staining similar to Descemet 2016-87-IM-15. The corneal scrapings of patients with clinically membrane (DM) was called 4+. suspected microbial keratitis, processed in the microbiology department as per our institute protocol,24 were included. Thirty- seven nonconsecutive blocks of FFPE ocular tissues [32 half Validation of IKI-H2SO4 Staining Techniques corneal buttons (CBs) and 5 evisceration specimens] of 36 on Corneal Scrapes and Pure Cultures patients were collected, for which the species of the infective In addition, slides of 7 corneal scrapings stained pre- organism was already identified and confirmed in the microbi- viously with potassium hydroxide-calcofluor white (KOH- ology department. Fourteen of these FFPE tissues (10 CBs and 4 CFW) and Gram stain and diagnosed as having either fungi eviscerations) of 13 patients had microbiological diagnoses of or Pythium (referred as SCR 01–07 on morphology alone) were Pythium insidiosum, based on the morphology and zoospore also retrieved. These slides were destained, hydrated, and formation, and confirmed by ITS DNA sequencing. Twenty- subjected to IKI solution (iodine concentration: 0.4 g) for 2 three of the tissues (22 CBs and 1 evisceration) were cases of hours followed by 40 mLof65%HSO and observed under fungal keratitis and served as controls. These included Asper- 2 4 a microscope after placing a cover slip. These cases were also gillus spp.(10),Fusarium spp.(8),Cladosporium (2), and one studied for growth in culture media [blood agar, chocolate agar, each of Acremonium, Candida,andColletotrichum.Thecases and Sabouraud dextrose agar (SDA)]. Samples that did not of fungal keratitis that had resolved with medical therapy were grow on SDA but showed typical colonies on chocolate agar or not included. Gastrointestinal tissue of one case of Mucormy- blood agar were studied for zoospore formation in the cosis was also included in the study. Adequacy and the presence induction medium, using carnation leaf. Smears of pure of the infective organism in all the paraffinblockswere cultures (from SDA/blood agar/chocolate agar) of these 7 confirmed using hematoxylin–eosin (H&E) stain and Gomori cases were also studied by staining with IKI solution for 2 methenamine silver (GMS) stain, and all the slides were re- hours and 65% H SO . viewed by 2 senior pathologists (R.M. and S.A.). Sections for 2 4 staining were deparaffinized by heating at 55 to 60°C for a duration of 20 minutes followed by immersing in xylene (15 Validation of the Staining Technique on dips)-3 changes, subsequently rehydrated using graded alcohol Archived Destained H&E Slides and washing in water. Iodine–potassium iodide–sulfuric acid Archived slides of 10/13 cases of microbiologically stain (IKI-H SO ) and periodic acid–Schiff (PAS) stain with 2 4 diagnosed Pythium keratitis, previously