Pythium Insidiosumkeratitis: Clinical Profile and Role of DNA

CLINICAL SCIENCE

Pythium insidiosum Keratitis: Clinical Proﬁle and Role of DNA Sequencing and Zoospore Formation in Diagnosis

Savitri Sharma, MD,* Praveen K. Balne, MSc,* Swapna R. Motukupally, MD,* Sujata Das, MS, FRCS(Glasg),† Prashant Garg, MS,‡ Srikant K. Sahu, MS,† Kotakonda Arunasri, MSc,§ Kodiganti Manjulatha, MSc,* Dilip K. Mishra, MD,¶ and Sisinthy Shivaji, PhD*

Conclusions: P. insidiosum keratitis needs to be considered in the Purpose: To report the molecular and microbiological diagnosis differential diagnosis of severe fungal keratitis. It can be identified fi and clinical pro le of 13 patients with Pythium insidiosum keratitis. using the zoospore formation method and confirmed by ITS DNA Methods: Phase 1 of the study consisted of DNA sequencing of the sequencing. Lack of response to currently used antifungal drugs calls ITS region of the rDNA of 162 stocked morphologically unidentified for evaluation of newer drugs for medical therapy and consideration nonconsecutive fungal isolates from corneal scraping of patients for early penetrating keratoplasty. – with keratitis (2010 2012). Blast and phylogenetic analyses of the Key Words: Pythium insidiosum, keratitis, diagnosis, treatment, sequences showed 9 to be P. insidiosum. A retrospective review of zoospore, DNA sequencing archived photographs of colony and direct microscopy of corneal scrapings and clinical records of the cases were performed. Phase 2 (Cornea 2015;34:438–442) 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. phthalmic literature abounds with information on all Oaspects of fungal keratitis.1 Correlation of etiological Results: The prevalence of P. insidiosum among unidentified agent to treatment outcome provides a logical ground to fungal isolates from keratitis was 9/162 (5.5%) in phase 1. In phase develop strategies for treatment. A large number of fungal 2, 4/102 cases (3.9%) of fungal keratitis were identified as species are known to cause keratitis; however, many studies P. insidiosum (January–February, 2014). Phylogenetic analysis of have reported 10% to 23% of fungal isolates from patients all 13 fungal isolates confirmed the identification of P. insidiosum. with fungal keratitis as unidentified because of lack of Corneal infiltrates exhibited hyphate edges, tentacle-like extensions, sporulation in culture.1–3 Molecular microbiologists have been and dot-like infiltrates surrounding the main infiltrate. Response to quick to adopt DNA sequence analysis to unravel the identity topical 5% natamycin eye drops with or without oral antifungals was of nonsporulating fungi.4 poor (penetrating keratoplasty: 9 and evisceration: 2) with a mean Analysis of our data spanning over a decade (1991– follow-up period of 82 days. 2000) showed that 10% to 13% of fungal isolates were morphologically unidentifiable because of lack of sporulation.2 Received for publication June 13, 2014; revision received November 11, DNA sequencing of such isolates revealed the presence of 2014; accepted November 12, 2014. Published online ahead of print Pythium insidiosum among them. A retrograde archival search March 2015. for direct microscopic and colony morphology pictures of these From the *Jhaveri Microbiology Centre, Hyderabad Eye Research Foundation, cases led to the realization that the isolate was not uncommon. L. V. Prasad Eye Institute, Hyderabad, India; †Cornea and Anterior fi Segment Services, Hyderabad Eye Research Foundation, L. V. Prasad Eye We report the clinical and microbiological pro le of 13 Institute, Bhubaneswar, India; ‡Cornea and Anterior Segment Services, nonconsecutive cases of P. insidiosum keratitis along with Hyderabad Eye Research Foundation, L. V. Prasad Eye Institute, the phylogenetic analysis and a simple method of zoospore Hyderabad, India; §Indian Institute of Chemical Technology, Hyderabad, demonstration in the laboratory that aids in the detection of this India; and ¶Ophthalmic Pathology Service, Hyderabad Eye Research underreported etiological agent of fungal keratitis. Foundation, L. V. Prasad Eye Institute, Hyderabad, India. 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 MATERIALS AND METHODS by a senior research fellowship from the Council of ScientificandIndustrial The study was conducted at L. V. Prasad Eye Institute, Research (CSIR), Government of India, New Delhi, India. India. The institutional protocol requires complete clinical The authors have no conflicts of interest to disclose. Supplemental digital content is available for this article. Direct URL citations and microbiological investigation of all patients presenting appear in the printed text and are provided in the HTML and PDF with suspected microbial keratitis in the cornea clinics of the versions of this article on the journal’s Web site (www.corneajrnl.com). institute. Our microbiological procedures published earlier Reprints: Savitri Sharma, MD, Jhaveri Microbiology Centre, Brien Holden have described the criteria to determine the significance of Eye Research Centre, Hyderabad Eye Research Foundation, L. V. Prasad 2 – Eye Institute, Rd No. 2, Banjara Hills, Hyderabad 500034, India (e-mail: culture isolates. In phase 1 (2010 2012) of the study [email protected]). (approved by the institutional review board of L. V. Prasad Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. Eye Institute, Hyderabad, reference: LEC 08046, dated April

438 | www.corneajrnl.com Cornea Volume 34, Number 4, April 2015

27, 2009), 162 unidentified fungal isolates from corneal et al7 with a modification (carnation leaf, incubation in an scraping of patients with fungal keratitis were stocked and induction medium for 24 hours). These additional 4 fungal were analyzed in 2013. DNA was isolated using QIAamp isolates were also subjected to sequencing as described DNA kit (Qiagen, Germany), and ITS1-5.8S-ITS2 regions of before. Clinical data of all 13 patients, identified to have the rRNA gene were amplified by PCR as described earlier,5 P. insidiosum keratitis in 2 phases of the study, were analyzed and the purified PCR products were sequenced using the same retrospectively. Topical 5% natamycin eye drops with or primers. The sequences that were identified as P. insidiosum without systemic antifungals were used for treatment of all were aligned using the CLUSTAL W algorithm, and the patients. Based on the response to medical therapy, surgical length was manually corrected to 450 bp. Phylogenetic trees treatment included therapeutic penetrating keratoplasty (PK) were constructed using the neighborhood joining method or evisceration. Corneal buttons were submitted for histopa- available in MEGA6.6 Maximum likelihood and maximum thology and microbiological evaluation.2 parsimony algorithms were also used. Archived clinical and microbiology records and photographs were retrieved for 9 isolates whose sequence type was RESULTS P. insidiosum. The microscopic and culture characteristics Demographic, clinical, and laboratory findings of all were noted. In phase 2, between January 7, 2014, and patients (9 from phase 1 and 4 from phase 2) are shown in February 5, 2014, corneal scrapings (4 patients) that demon- Table 1. The prevalence of P. insidiosum as an etiological strated characteristic colony and microscopic features of agent of fungal keratitis was 5.5% (9/162) in phase 1 and P. insidiosum were studied. All 4 cultures were tested for 3.9% (4/102) in phase 2. All 13 fungal isolates were cultured zoospores according to the method described by Mendoza from corneal scrapings or corneal buttons. Although all were

TABLE 1. Demographic, Clinical, and Microbiological Features of 13 Patients With P. insidiosum Keratitis Corneal Scraping Patient No. Accession No. Age (yrs) Gender Occupation Risk Factor KOH + CFW Gram Stain Culture* 1 JX868641 49 Male Farmer Grass No Org. FF UHF 2 JX868664 28 Male NA Smoke FF FF UHF 3 JX868706 59 Male NA NA FF FF UHF 4 JX868709 33 Male Business Stick FF FF No growth 5 JX868764 47 Male Farmer Sand FF FF UHF + S. aureus 6 JX868765 64 Male NA Insect FF FF UHF 7 JX868766 49 Male NA None No Org. GPC S. pneumoniae 8 JX868767 51 Male Farmer None No Org. No Org. No growth 9 JX868769 51 Male Farmer Leaf FF FF UHF 10 KJ584656 46 Male Farmer Grass FF FF P. insidiosum 11 KJ584657 33 Male Farmer None FF FF P. insidiosum 12 KJ584658 46 Male Farmer None FF FF P. insidiosum 13 KJ584659 44 Female NA None FF FF P. insidiosum

Visual Acuity Treatment Patient No. At Presentation Final Medical Surgical Corneal Button Culture Duration of Follow-up (d) 1 LP LP Nata, Keto PK UHF 88 2 LP LP Nata, Itra PK ND 122 3 NR LTFU Nata None LTFU (ND) LTFU 4 20/400 20/50 Nata PK UHF 137 5 LP 20/80 Nata, Gatiﬂox PK (Twice) UHF + S. aureus 470 6 LP NAp Nata, Keto Evisceration ND 40 7 LP LP Gatiﬂox PK UHF 19 8 LP NAp Nata PK, Evisceration UHF 23 9 HM LP Nata, Itra None LTFU (ND) 10 10 HM CFCF Nata PK P. insidiosum 35 11 CFCF 20/160 Nata, Keto PK P. insidiosum 63 12 LP CF 1.5 m Nata PK NG 39 13 HM CF 1.5 m Nata, Keto PK P. insidiosum 28

*Blood agar at 37°C. CFCF, counting finger close to face; FF, fungal filaments; GPC, gram-positive cocci; HM, hand movement; Itra, itraconazole tablet; Keto, ketoconazole tablet; LP, light perception; LTFU, lost to follow-up; NA, not available; NAp, not applicable; Nata, 5% natamycin eye drop; ND, not done; NG, no growth; NR, not recorded; Org., organisms; UHF, unidentified hyaline fungus.

confirmed by rDNA sequencing, zoospore formation was varied from 7.5 mm2 to total corneal infiltrate, and patients demonstrated in fungal isolates from patients 10 to 13 in had presented between 4 days to 2 months from the onset of phase 2. The typical sporangium with bursting out of highly symptoms. The visual acuity at the time of presentation ranged motile zoospores and rapid migration of zoospores through from 20/400 to light perception. A predisposing factor was the sporangiophore can be seen in Supplemental Digital elicited in 7/13 (53.8%) patients (Table 1). The majority of the Contents 1 and 2 (see Videos, http://links.lww.com/ICO/ patients were farmers. Although hyphate edges of the infiltrates A258 and http://links.lww.com/ICO/A259). Zoospore forma- were seen in most patients, slit-lamp biomicroscopy showed tion for isolates 1 to 9 in phase 1 was not studied because the multiple linear tentacle-like infiltrates (Fig. 2A, patient 11) in 3 culture isolates were not available after sequencing. of 13 patients and the presence of dot-like infiltrates (Fig. 2B, Of 13 sequences identified as P. insidiosum, sequences patient 12) at the midstromal level surrounding the main infiltrate of 10 isolates were selected (based on the aligned length in in 4 of 13 patients. Hypopyon was present in 6 of 13 patients. the ITS2 region) for phylogenetic analysis using bioinfor- None of the patients responded to medical therapy, and matics software. In the Neighbor joining tree, the all patients were advised to have therapeutic PK. Broad P. insidiosum isolates from this study were confirmed on fungal elements were demonstrated in paraffin sections of the the basis of formation of robust clades with several isolates corneal buttons (Fig. 1D, patient 10) in all 9, and culture grew from other hosts along with the P. insidiosum type strain typical colonies in 8 of 9 cases. The mean follow-up period of available in the NCBI GenBank database (see Supplemental the patients was 82 days, and the final visual outcome was Digital Content 3, http://links.lww.com/ICO/A260). The poor in all patients except 2 (Table 1). identification was also confirmed by maximum likelihood and maximum parsimony analyses of the strains. All but 2 patients were male, and the average age of the DISCUSSION patients was 46.1 years (Table 1). Sparsely septate, relatively P. insidiosum is a fungus-like microbe that morpholog- broad, ribbon-like fungal filaments, as shown in Figure 1A, ically exhibits features of branching, sparsely septate or were detected in corneal scrapings by direct microscopy in aseptate filaments. It is classified as an oomycete and lacks 84% of the patients (10% potassium hydroxide with 0.1% ergosterol in the cytoplasmic membrane. Unlike fungi, chitin calcofluor white 277% and Gram stain 284%). Flat, feathery- is not the main compound in the cell wall, and asexual edged, partially submerged, glabrous, colorless or light brown reproduction in the oomycetes occurs in the form of sporangia colonies on blood agar were grown from corneal scrapings or containing biflagellate zoospores.8 buttons (Fig. 1B). The clinical slit-lamp image of patient 1 is In this presentation, we wish to bring forth to the notice shown in Figure 1C. The clinical diagnosis in all patients was of ophthalmologists and ocular microbiologists alike that fungal keratitis. The stromal infiltrate size at presentation although rarely reported, P. insidiosum keratitis may not be

FIGURE 1. Clinical and laboratory findings of patients with P. insidiosum keratitis. A, Broad, aseptate, branching at right angles, fungal filaments in corneal scraping (patient 5, 10% potassium hydroxide with 0.1% calcofluor white stain, ·400). B, Flat, feathery, colorless colony on 5% sheep blood agar (patient no. 5, 24 hours, 37˚C). C, Slit-lamp picture of the cornea of a 49-year-old patient (patient 1) who presented with symptoms of 3 weeks of duration to us with central stromal infiltrates. The infiltrate is denser in periphery: (D) broad, sparsely septate, fungal elements in the corneal stroma (patient 10, Gomori methenamine silver stain, ·400).

FIGURE 2. Slit-lamp image of the cornea under diffuse illumination showing (A) inferior stromal infiltrates with multiple linear tentacle-like infiltrates extending from the superior edge up to the limbus (patient 11) and (B) dry, full-thickness infiltrates with hyphate edges and multiple dot-like infiltrates surrounding the main infiltrate (patient 12).

a rare infection. It simply goes unrecognized. Although the usually been trauma to the cornea or contact lens wear.9,15 As majority of the reports on human pythiosis including keratitis has been observed earlier,16 there are no characteristic clinical are from Thailand, the disease has been reported from other features of P. insidiosum keratitis. This study found the parts of the world.9–11 Once we became familiar with the infection to be more common in males and young adults, microscopic and colony morphology of P. insidiosum and which is similar to fungal keratitis in general.3 The reticular learnt to demonstrate the formation of zoospores, we could pattern of subepithelial and superficial infiltration described by identify 4 cases in 1 month. Thanathanee et al17 was not seen in any of our patients. Efforts A retrospective analysis of clinical features did not to recognize this fungus in the laboratory need to be matched show pathognomonic features to help make a clinical diag- with studies to describe typical clinical features, which will nosis; however, tentacle-like or dot-like corneal infiltrates help make a preliminary clinical diagnosis. In vitro and in vivo may be notable features. Well-designed prospective studies of studies to determine appropriate treatment for P. insidiosum clinical features on a larger group of patients may determine keratitis are urgently needed because the conventional anti- the sensitivity and specificity of characteristic clinical features fungal agents (eg, polyenes, azoles) are not effective. P. in making a clinical diagnosis. insidiosum vaccine, shown to be effective to prevent recurrent Demonstration of zoospores is simple and takes 2 days systemic infections in patients with thalassemia, has been used after the fungus has grown on blood agar (24–48 hours), to prevent postkeratoplasty recurrence in corneal grafts.17 which is similar to the time that may be taken to perform Nevertheless, these authors remained unsure of the efficacy sequencing, but several times less expensive and relatively of the vaccine and attributed the lack of recurrence in their low on skill demand. We recommend routine adoption of this cases to early keratoplasty with a wide surgical excision. technique in ocular microbiology laboratories for all typical Studies have been published that evaluated single and colonies growing on blood or chocolate agar at 37°C with no combination antifungal drugs with an alternative mechanism growth or poor growth on Sabouraud dextrose agar at 25°C. of action,18 and it is hoped that some of these will find their Zoospore formation is most commonly seen in P. insidiosum, way to effective medical treatment of P. insidiosum keratitis. although other species of oomycetes such as Phytophthora and Lagenidium are known to produce zoospores. Until REFERENCES recently, P. insidiosum was the only pathogenic species in 12 1. Thomas PA. Current perspectives on ophthalmic mycoses. Clin Micro- humans. However, in 2011, Pythium aphanidermatum was biol Rev. 2003;16:730–797. 13 reported to cause wound infection after combat trauma. No 2. 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