FP679 : Pythium Keratits – Clinical Profile and Measures To Reduce Recurrence. Dr. Shweta Agarwal

A12280, Dr. Bhaskar Srinivasan, Dr. Mamta Agarwal, Dr. Geetha Iyer

Pythium keratitis- Clinical Profile And Measures To Reduce Recurrence

Shweta Agarwal

Geetha Iyer

Bhaskar Srinivasan

Mamta Agarwal

Lily Therese

CJ Shah Cornea Services / Dr G Sitalakshmi Memorial Clinic for Ocular Surface Disorders

Medical Research Foundation, Sankara Nethralaya, 18 College Road, Chennai – 600006, Tamil Nadu, India.

Corresponding author:

Dr Geetha Iyer

CJ Shah Cornea Services

Dr G Sitalakshmi Memorial Clinic for Ocular Surface Disorders

Medical Research Foundation, Sankara Nethralaya

18, College Road, Chennai -600 006

Phone: 044-28271616, Fax: 044-28254180, Email id: drgeethaiyer@gmail.com 


Aim: To report clinical profile of pythium keratitis and measures to reduce recurrence.

Method: Retrospective study of 10 patients with Pythium keratitis.

Results: Slit lamp findings revealed central 4-7mm dense infiltrate with radiating superficial reticular pattern.Based on PCR based DNA sequencing confirmation of Pythium insidiosum as the causative organism, subsequent therapeutic penetrating keratoplasty was combined with inraop cryotherapy at the host edge. Recurrence was noted in 1/5 eyes post cryotherapy. 8/10 eyes were anatomically salvaged with a follow up ranging from 1-24 months.

Conclusion: Pythium clinically mimics fungal keratitis and early surgical intervention is the only treatment option.Intraop cryotherapy might help reduce recurrence.

 Pythium keratitis- Clinical Profile And Measures To Reduce Recurrence


Pythium insidiosum causes diseases in humans and animals, mostly in tropical, subtropical and temperate countries. It is classified in the Phylum Straminipila, Class Oomycetes, Order Pythiales and Family Pythiaceae. [1] Oomycetes have a cell wall consisting of cellulosic compounds and glycan and not chitin or ergosterol, features which distinguish them from fungus. Though corneal infections caused by Pythium insidiosum are rare they could lead to significant morbidity and globe threatening complications. [2-5]

Reported management options for Pythium keratitis have been antifungal agents, extensive debridement and immunotherapy with P insidiosum vaccine.Retrospective studies from India have described the microbiological characteristics of the organism isolated from patients with microbial keratitis and correlated it with the clinical features.

In this paper we report the clinical profile and measures to reduce recurrence of Pythiuminsidiosum keratitis managed at a tertiary eye care centre following PCR identification of the same.This series highlights the need to be aware of this clinical entity which clinically mimicks refractory fungal keratitis, incidence of which is very high in a tropical country like India and at the same time suggests options to improve outcomes. 


The Institutional Review Board approval was obtained for this study which adhered to the tenets of the Declaration of Helsinki. Case records of 10 patients diagnosed to have pythium keratitis between April 2014 and Feburary 2015 were analyzed retrospectively.Routine corneal scraping for Gram’s and KOH smear was performed. The infective material from corneal scraping or from the excised corneal button following therapeutic penetrating keratoplasty was cultured on blood agar, chocolate agar and Sabourad’s dextrose medium and was processed for species identification. In these 10 eyes, septate fungal filaments were noted on KOH smear, and the filamentous fungus could not be identified by conventional method. Cultures revealed the growth of a flat non-sporulating mold. PCR based DNA sequencing targeting ITS region identified the organism as Pythium insidiosum.[6] The antifungal drug sensitivity was performed by agar dilution method to routinely used antifungals[7] (voriconazole, amphotericin B, fluconazole and itraconazole)  and revealed resistance to all.The isolate was also subjected to indigenously designed sensitivity testing for polyhexamethylenebiguanide (PHMB). It was found to be sensitive to PHMB > 0.01% and 0.02% concentration by agar dilution method (indigenously standardized method).

All eyes underwent a therapeutic penetrating keratoplasty (PK) either because of evidence of worsening or if they presented with limbus to limbus involvement or following PCR confirmation of the causative organism whichever was earlier. Postoperatively all eyes were started on PHMB 0.02% 1 hourly and voriconazole 1% 1 hourly for a minimum period of 3 weeks. A repeat therapeutic PK was performed at the earliest sign of recurrence .Recurrence was noted in 8/10 eyes and 5/8 eyes were subjected to a single freeze thaw cryotherapy to the host edge of the trephined mark following conjunctival peritomy prior to entering the eye.Post cryotherapy, an annular area of 2-3 mm beyond the trephine mark was swabbed using absolute alcohol in 2 eyes.


10 patients were diagnosed with Pythium keratitis using the PCR technique for identification in a period of 11 months. Of the 10 patients, 2 were females, with age ranging from 19 years to 50 years. Only 3 were farmers by occupation, 5 were software professionals and 2 were housewives from urban locales with no exposure to vegetable matter. Only 1 patient gave a history of exposure to water in swimming pool. All others gave a probable history of some dust particle entering the eye followed by initiation of symptoms. 4 eyes presented with a limbus to limbus infiltration with corneal melt, while the remaining 6 eyes showed a central 4- 7 mm infiltration with radiating superficial reticular pattern involvement. All patients were on topical antifungal agents on presentation. All patients revealed septate fungal filaments on KOH and were being treated with topical antifungals, natamycin 5 % eye drop 1 hourly and voriconazole 1% eye drop 1 hourly. All patients underwent the 1st therapeutic PK based on worsening or non responsiveness of clinical features to the antifungal regimen. Following the growth of a non-sporulating mold in the 1st patient with an aggressive recurrence after surgical intervention, the infective material was subjected to PCR identification for P insidiosum due to a high index of suspicion and confirmed.  Subsequently all patients exhibiting growth of non-sporulating mold were subjected to PCR identification. Remaining 9 patients also revealed P insidiosum as the causative organism. In all patients the identification was revealed after the 1st therapeutic penetrating keratoplasty (TPK) and further surgical interventions were carried out at the earliest sign of recurrence. 8/10 eyes had recurrence of which 2 eyes underwent evisceration. 5/8 eyes with recurrence underwent single freeze thaw cryotherapy to the host edge prior to entering the eye at the time of re-TPK. 6 eyes were phakic at the end of all surgical interventions. The corneal graft size at the time of last therapeutic PK ranged from 11 mm to 12.5 mm. In 1 eye, due to the adjacent scleral involvement extending to 14 mm, a lamellar scleral dissection to remove the infected sclera was done. This was followed by cryotherapy and application of absolute alcohol to the bed. A large 16 mm donor graft was trephined. The peripheral scleral rim was dissected to reduce its thickness. The infected cornea with a partial thickness scleral frill was excised from the host. The 16 mm fashioned donor graft was secured peripherally to the sclera using fibrin glue and 4 sutures were taken in each quadrant at the limbus to anchor the donor cornea to the residual host sclera.   The mean follow-up was 13months (1-24 months) excluding those who underwent evisceration. Of these 8 eyes, 2 underwent optical PK and 1 is waiting. 


 In tropical and subtropical countries where fungal corneal infections predominate, awareness regarding an organism that mimics fungal keratitis clinically as well as microbiologically and is resistant to routine antifungals becomes very important. Reported management options for pythium is mainly by extensive debridement, antifungal agents, [2] immunotherapy, [2] combination of allylamines (terbinafine) with amphotericin B, fluconazole or caspofungin, [8] photodynamic therapy [9]   and have all been tried with minimal efficacy. Therapeutic PK, removing the infected tissue has to be performed at the earliest. Not only it’s resistance to antifungals, but its propensity to recur with aggressiveness, further complicates the management. Evisceration has been resorted to in eyes with multiple recurrences.[2]

Noting of filamentous fungus on KOH smear, the growth of nonsporulating mold on culture media, the aggressive recurrence of infection and prior reports regarding Pythium keratitis in a similar setting, raised a high index of suspicion in the 1st patient that led to confirmation of the organism by DNA sequencing. Culture identification with zoospore induction can be used to confirm the organism and 2 different techniques have been described. Though a recent modification suggests reduced time taken for identification from 5-7 days to 2 days, the authors also mention that DNA sequencing is required for confirmation of the species.

Our study noted the infection to be seen in healthy young to middle-aged individuals. Contrary to the high incidence of fungal/Pythium infection usually noted among farmers, most patients in this study were software professionals and/or housewives with no exposure to vegetable matter or water. Thalassemia was noted to be a predisposing factor among patients with Pythium keratitis from Thailand; however the same was not noted in our patients. The reticular pattern of infiltration extending towards the limbus from a dense central stromal infiltrate documented by Thanathanee et al was seen in 6 eyes in our study, necessitating a large corneal graft in all these eyes.

In all, of the 45 eyes reported in literatureas pythium keratitis, 18 (40 %) required evisceration, 23 required single or multiple grafts and 3 were lost to follow-up. In our series of 10 eyes, 2 (20%) were eviscerated, while the remaining 8 patients are being followed up over a mean period of 12 months with an optical keratoplastybeing done for 2patients.5/8 eyes underwent cryotherapy during re -therapeutic PK with only 1 developing a recurrence. The extent of the corneal infiltration did not seem to have a bearing on the outcome. All eyes with a total corneal infiltrate were salvaged. Whether this is related to the virulence of the organism is not known. Specific measures were taken to preserve the crystalline lens during each intervention, irrespective of the graft size to prevent the entry of the organism into the posterior segment. Glaucoma was anticipated and was noted in 4 out of the 6 eyes and was managed medically

In addition to surgical intervention, PHMB was instituted in all eyes. Indigenously designed antifungal sensitivity testing for PHMB (0.01and 0.02% by agar dilution method) was conducted which revealed sensitivity to PHMB. This was done considering the presence of cellulose in the cell walls of both pythium and acanthamoebae, for which PHMB is routinely used. Despite the use of PHMB, recurrence was noted in all eyes. The additive role of PHMB in the management of pythium keratitis is yet to be ascertained.

It is known that despite being the only management measure for pythium keratitis, the outcome of surgical therapeutic PK is poor with a high risk of recurrence. In view of this, with no other established modality of treatment, we combined the keratoplasty with application of single freeze thaw cryotherapy to the host edge. This was done as an adjunctive measure to kill the organisms, if any, along the host edge and thereby reduce the risk of recurrence.Rapid cooling during cryotherapy can form intracellular ice crystals causing cell disruption and the rewarming/thawing can damage the cell membrane again.11 In 2 cases, following cryotherapy, absolute alcohol was applied along the same area. Ethanol has been found to decrease the viability of cells by causing cell lysis, suppressing proliferation and inducing apoptosis.12 For an organism that has no known medical management, with a poor prognosis following surgical intervention, intraoperative cryotherapy and use of absolute alcohol could constitute adjunctive measures to reduce the risk of recurrence.

The aims of this paper are many. Primarily, it is to highlight the need to be aware of the possibility of Pythium insidiosum keratitis in apparently refractory fungal keratitis in India.The paper highlights the occurrence of the infection in the non-farmer population, in the absence of any specific inciting factor. Apart from this, means to early identification by inducing zoosporogenesis or using PCR DNA sequencing analysis should be done in cases with a high degree of suspicion to hasten the diagnosis and thereby intervention. Since surgery is the only option in these eyes, it has to be done at the earliest to improve outcomes. Also since recurrences are common, adjunctive cryotherapy and / or absolute alcohol could be used in an attempt to reduce the same.

We therefore reiterate the need for being aware of Pythium insidiosum keratitis as an entity and the need for its early identification and aggressive surgical measures to salvage the eye from an otherwise devastating ocular infection, especially in India, where fungal corneal infections predominate.


1.Thomas PA. Current perspectives on ophthalmic mycoses.ClinMicrobiol Rev.2003; 16: 730-797.

2.Chakrabarti A, Singh R. The emerging epidemiology of mould infections in developing countries.CurrOpin Infect Dis. 2011 Dec; 24(6):521-6.

3.Onsiri T, Orapin E, Ram R, et al. Outbreak of pythium keratitis during rainy season: a case series. Cornea 2013; 32: 199-204.

4.Badenoch PR, Mills RA, Chang JH, et al. Pythium insidiosum keratitis in an Australian child. Clin Experiment Ophthalmol.2009; 37: 806-809.

5.Lekhanont K, Chuckpaiwong V, ChongtrakoolP, et al. Pythium insidiosum keratitis in contact lens wear: a case report. Cornea 2009; 28: 1173-1177.

6.BagayalakshmiR, Therese KL, Prasanna S, et al. Newer emerging pathogens of ocular non-sporulating molds identified by polymerase chain reaction (PCR) – based DNA sequencing technique targeting internal transcribed spacer (ITS) region. Curr Eye Res.2008; 33: 139-147.

7.Therese KL. Bagyalakshmi R., Madhavan H.N. Deepa P. In – Vitro antifungal susceptibility    testing by agar dilution method to determine the minimum inhibitory concentrations of   Amphotericin B, Flucanozole and ketaconazole against ocular fungal isolates. Indian Journal of Medical Microbiology 2006.     24(4):273-9

8.CavalheiroAS, MaboniG, deAzevedoMI, etal.Invitro activity of terbinafine combined with capsofungin and azoles against Pythium insidiosum. Antimicrob Agents Chemother.2009; 53: 2136-2138.

9.Layla P, Sandra B, Mauricio B, et al. Photodynamic therapy in Pythium insidiosum- An invitro study of the correlation of sensitizer localization and cell death. Plos one. 2014; 9

10.Anshu A, Parthasarathy A, Mehta JS, et al. Outcomes of therapeutic deep lamellar keratoplasty and penetrating keratoplasty for advanced infectious keratitis: a comparative study. Ophthalmology 2009, 116: 615-23.

11.Yingxin C, WeigiaY, Minghong G, et al.Experimental study on cryotherapy for fungal corneal ulcer.BMC ophthalmology (2015); 15:29

12.JooYoun O, Ji Min Y, Jung Hua K. Analysis of ethanol effects on corneal epithelium.IOVS (2013)  ;54:3852-3856

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