Colletotrichum graminicola keratitis: First case report from India

Colletotrichum graminicola is a medically important fungus belonging to the order Melanconiales under the class Coelomycetes. The members of the genus Colletotrichum are primarily plant pathogens which cause anthracnoses (fungal infection in plants). In the past few decades, they are progressively being implicated as etiological agents of subcutaneous hyalohyphomycoses and keratomycoses. Of the five medically important members in the genus Colletotrichum, keratitis due to Colletotrichum graminicola is rare. We diagnosed Colletotrichum graminicola keratitis in a 44-year-old man who presented with a non-healing corneal ulcer since three weeks. Positive smears and cultures from the corneal scrapings established the causative organism as C. graminicola. The patient was treated with a combination of oral ketoconazole and topical fluconazole and natamycin. Infection resolved over 10 weeks and antimicrobials were stopped. We describe the clinical presentation and treatment outcome of Colletotrichum graminicola keratitis.

to our eye department with a history of pain, redness and watering in the right eye of 25 days duration. There was no history of trauma. He had initially been seen by a local ophthalmologist, who treated him with ciprofloxacin eye drops, cycloplegics, and acyclovir ointment. His symptoms did not subside, so he was referred to our institute for further management. On presentation, the best-corrected visual acuity in his right eye was 20/500. On examination, he was found to have a corneal ulcer with infiltrate measuring 6 x 4 mm in size and epithelial defect of 6 mm involving the temporal half of the cornea [ Fig. 1]. There was no hypopyon, satellite lesions or endothelial plaque. Anterior chamber showed Grade 3 cells.
Corneal scrapes were obtained from the active edges and smears were sent for staining with Gram, Giemsa and potassium hydroxide (KOH). Material was inoculated onto plates for bacterial, fungal and Acanthamoeba culture. Sabouraud's dextrose agar (SDA) and sheep blood agar (SBA) were incubated at 28 0 C and 37 0 C, respectively. For culturing Acanthamoeba, non-nutrient agar with Escherichia coli overlay was employed and incubated at ambient temperature. Smears revealed fungal filaments [ Fig. 2]. After two days, filamentous fungi was grown on SDA; gradually, at the end of two weeks, the colony assumed a salmon color with numerous black sclerotia [ Fig. 3] and an orange color on the reverse that later became dark brown. A lactophenol cotton blue tease mount preparation from the colonies showed abundant setae [ Fig. 4] wide, falcate, fusiform conidia gradually tapering at the apex and base and abundant appressoria with irregular margins. The fungus was initially identified as Colletotrichum graminicola and further confirmation carried out at the Centraalbureau voor Schimmelcultures (CBS), Fungal Biodiversity Centre, Utrecht, Netherlands. Antifungal susceptibility testing for the isolate was performed using the Clinical and Laboratory Standards Institute Document M38-A2. The minimum inhibitory concentration (expressed as microgram per milliliter) for amphotericin, itraconazole and ketaconazole at 48 h and 72 h was found to be 0.5/1, 0.5/1 and 1/2 respectively. SBA was sterile after 48 h of aerobic incubation and there was no culture recovery of Acanthamoeba.
Treatment was started with oral ketoconazole 200 mg twice daily (liver functions were normal) which was continued for two weeks. Fluconazole 0.3% eye drops, natamycin 5% eye drops were given hourly and atropine 1% eye drops was used thrice a day. Epithelial defect healed by six weeks and the infiltrates fully resolved by eight weeks [Fig. 5]. After 10 weeks, all medications were tapered and stopped. At 21 weeks, patient had a corneal opacity and the unaided corrected visual acuity was 20/30 and N6.

Discussion
Colletotrichum is a ubiquitous fungus with a well-documented phytopathogenic potential. It is most frequently isolated from soil and plant vegetation, and has subtle morphological features making identification in culture difficult. The falcate conidia can be confused with Fusarium spp., a common agent of keratomycosis. Among the five medically important Colletotrichum species, falcate conidia are present only in C. dematium and C. graminicola. The characteristic presence of 4-6 µm-wide conidia and irregular margins of appressoria seen in C. graminicola helps in easy delineation of the species from C.

Indian Journal of Ophthalmology
Vol. 58 No. 5    dematium, which has a 3-4 µm-wide (narrower) conidia and smooth margins of appressoria. [3] SDA can be used for primary isolation of Colletotrichum from corneal scrapes. To enhance appresoria and sclerotia formation water agar with added plant tissue, carnation leaf agar and oatmeal agar have been recommended. [3] In the literature, the principal Colletotrichum species implicated in keratomycosis has been C. dematium along with a documented report of C. gleosporoides. [6][7][8][9] In most instances, speciation of isolated Colletotrichum species was not attempted; hence the exact frequency of isolation of C. graminicola remains obscure. Besides ocular trauma, insulin-dependent diabetes mellitus and prolonged use of corticosteroids have been reported as risk factors. [4,5] Our patient, had no such risk factors.
Earlier studies have reported complete resolution of Colletotrichum corneal ulcers with good visual recovery following treatment with natamycin, amphotericin B with azoles, 5 flucytosine with ciprofloxacin. [5] Ritterband et al. first reported keratitis due to C. graminicola that was difficult to treat and therapeutic penetrating keratoplasty was performed twice after which there was no recurrence of fungal infection. [10] In the present case we were able to treat successfully within eight weeks by a combination of azoles and natamycin. Based on the sensitivity report a combination of amphotericin B with azole and/or natamycin with dosing regimen extending 47 + 14 days is found to be effective in the therapeutic management of Colletotrichum keratitis.
Coleomycetous fungi belonging to the genus Colletotrichum spp. are opportunistic agents involved in keratomycoses. We report a rare case of C. graminicola keratitis. The report also highlights the successful treatment outcome of C. graminicola keratitis using a natamycin and azole combination.
Cycloposition (extent of ocular torsion) has been measured by various subjective methods including perimetry, double maddox rod test, Bagolini's glasses, indirect ophthalmoscopy lens, slit-lamp biomicroscopy and synoptophore. A reliable method to evaluate ocular torsion objectively is fundus photography. [1][2][3][4][5] However, wide variations have been reported in the measured results of the disc foveal angle (DFA) which is formed at the optic disc center between the horizontal meridian and the line joining the center of disc and foveal center. [2,3] DFA is indicative of the cycloposition of the eyes. The aim of the study was to evaluate the DFA and to find its correlation with possible influencing factors in children between 5-15 years of age.

Materials and Methods
A total of 105 children (210 eyes) were included in the study. The patients (and /parents) were briefed about the procedure and appropriate consent obtained for fundus photography. The age, sex and refractive errors of all patients were noted. We excluded all the patients with hazy media, abnormal muscle balance, retinal pathology and any manifest deviation. Photographic documentation of the ocular fundi of our patients was done by one of the authors (JP) using a TRC-50DX (Topcon, Japan) fundus camera taking care that the subject's head was well aligned -the side marks and chin rest were taken as a guide. Wide-field (50°) fundus photographs were taken. Children who were not cooperative or if the authors found were not able to keep their head straight were excluded from the study. Photographs were taken through the dilated pupil after instilling tropicamide 1% eye drops. The DFA was calculated from a well-focused single still photograph using IMAGEnet software (Topcon, Japan) and a protractor. To obtain the measurement of DFA, two lines were drawn; one straight line (horizontal meridian) passing through the center of the disc [ Fig. 1] (AD) and another line passing through the point D (center of the disc) and the Fovea (F) (DF). The angle between the fovea and the geometric centre of the disc (between the