Background

Fungal eye infections are among the main causes of visual disability in tropical and subtropical countries, such as India. Besides hyaline fungal species, which are responsible for the majority of keratomycoses and fungal endophthalmitis in the country, dark-pigmented fungi are also associated with emerging cases [1, 2]. The cosmopolitan genus Exophiala comprises dimorphic dematiaceous fungi commonly isolated from soil and plant debris [3, 4]. These fungi are the clinically most relevant black yeasts causing various uncommon forms of cutaneous, subcutaneous and disseminated (e.g., pneumonia and brain abscess) human infections [5], but eye infections due to Exophiala species are extremely rare. Most cases occur after a penetrating injury or post-eye surgery [6]. Though the presented case is the fourth documented case of endophthalmitis caused by E. dermatitidis across the globe according to a deep search of the available literature (Table 1) [7,8,9,10,11,12,13,14,15,16,17,18], it is unique, as it is the first report with a successful treatment outcome. Human ocular infection due to E. dermatitidis has not been documented in India before.

Table 1 A retrospective summary of Exophiala eye infections reported since 1990
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Case Presentation

A 59-year-old male patient presented at the Cornea Department of Aravind Eye Hospital and Postgraduate Institute of Ophthalmology (Coimbatore, Tamilnadu, India) in December 2012 with complaints of decreased vision, pain and redness in his left eye for the past 15 days. The patient gave a history of an uneventful cataract surgery with intraocular lens implantation in his left eye performed 4 months earlier. He had good vision in the first month following surgery, that is, visual acuity (VA) was 6/6. Three months later, he presented with gradual deterioration of vision (VA 6/60). The patient was otherwise in good health and had no known allergies.

On examination, the best corrected visual acuities were 6/6 in the right eye and 6/60 in the left eye. Intraocular pressure as measured by Goldmann applanation tonometry was 15 mm in the right eye and 13 mm in the left eye. Slit lamp examination of the right eye was within normal limits except for an early nuclear sclerotic cataract of grade 1. Examination of the left eye revealed circumcorneal congestion, a clear cornea and 3+ cells and 1+ flare in the anterior chamber. Texture and color of the iris were normal. The pupil was pharmacologically dilated and unresponsive to light. A posterior chamber intraocular lens was present, and a fluffy grayish white growth was noted on the posterior capsule (Fig. 1a, b).

Fig. 1
figure 1

Slit lamp biomicroscopy (a) with retro illumination (b) showing the fungal colonies on the posterior surface of the intraocular lens

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Indirect ophthalmoscopy of the right eye was within normal limits, but in the left eye moderate vitritis was observed. The patient underwent vitreous tap with a 24-gauge needle inserted into the central vitreous cavity along with intravitreal injections of moxifloxacin (500 µg/0.1 ml; Vigamox, Alcon Laboratories, USA) and voriconazole (100 µg/0.1 ml; Aurolab, India). He was placed on topical moxifloxacin (0.5% (w/v); Vigamox, Alcon, India) and fortified tobramycin (14 mg/ml; Toba, Sun Pharma Laboratories Ltd., India) eight times a day, plus voriconazole (1% (w/v); Vozole, Aurolab, India) eye drops six times a day and homatropine (2% (w/v); Homide, Warren, India) eye drops twice a day. The topical treatment continued till the time of the intraocular lens removal (1 month from the first vitreous tap).

Vitreous biopsies obtained during the procedure were plated on 5% sheep blood agar (SBA; HiMedia Laboratories, India), potato dextrose agar (PDA; HiMedia Laboratories, India) and thioglycolate broth (HiMedia Laboratories, India). A part of the sample was sent for molecular identification (Xcyton Diagnostics Limited, Bengaluru, India). Gram stained smears and 10% potassium hydroxide wet mount were also investigated. The smears as well as the cultures proved to be negative for fungal filaments. Molecular analyses were negative for all the tested bacteria and fungi. Postoperatively the same medications were continued. Prednisolone acetate (1% (w/v); Predforte, Allergan, India) was added at a frequency of six times a day. Dexamethasone (8 mg in 2 ml; Dexadran, Searle Labs Pvt. Ltd. India) was administered intravenously twice a day for 2 days and was subsequently replaced by oral prednisolone (40 mg/day; Omnacortil, MacLeods Pharmaceuticals Ltd., India), tapered over a period of 15 days.

On subsequent follow-ups, the visual acuity was gradually improving (i.e., VA 6/9 after 1 week post-vitreous tap), but the anterior chamber inflammation persisted, and fluffy deposits on posterior surface of the intraocular lens persisted. Oral steroids were discontinued, and the topical steroid dose (6 times per day; Predforte, Allergan, India) was tapered to four times. A repeated vitreous biopsy was performed on the 9th day following the first vitreous tap under local anesthesia using a 25-gauge vitreous cutter through the pars plana route. Care was taken to take the sample at the location of the fluffy growth with localized posterior capsulectomy and anterior vitrectomy. Specimens thus taken from the posterior capsule and anterior vitreous were plated on SBA, chocolate agar (CA; HiMedia Laboratories, India), PDA and Lowenstein Jensen medium (HiMedia Laboratories, India). A direct microscopic examination of Gram’s smears clearly revealed fungal filaments. The topical steroid therapy was stopped, and the frequency of topically applied voriconazole (1% (w/v)) was increased to hourly intervals. The therapy was supplemented with oral fluconazole (150 mg; Zocon, FDC Limited, India) twice a day. On the 5th day of incubation, a moderate-sized black, mucoid and yeast-like growth was noted on SBA, CA and PDA plates. The fungus was initially identified as E. dermatitidis based on colony morphology (Fig. 2a) and microscopic features (Fig. 2b) and was further subjected to molecular identification. Medical treatment was continued for the next 10 days. The infiltrate reappeared in the inferonasal quadrant in between the lens and the iris. Intraocular lens explantation and capsular bag removal with anterior vitrectomy were performed followed by an intravitreal voriconazole injection (100 µg/0.1 ml). Postoperatively, corneal edema and keratic precipitates were noted which resolved after 2 weeks. Intravitreal injections of voriconazole (100 µg/0.1 ml) were repeated twice in the ensuing week for persistent exudates in the inferior vitreous. While topical voriconazole (1% (w/v)) was continued and slowly tapered over 2 months, systemic fluconazole (150 mg twice daily) was maintained for a month. After a month, topical steroids (Predforte, Allergan, India) were started with a very low dose twice a day, and tapered very slowly over the next 4 months. The best corrected Snellen’s visual acuity with aphakic correction was 6/9.

Fig. 2
figure 2

Colony (a) and microscopic morphology (b) of Exophiala dermatitidis SZMC 21989 cultured on Sabouraud’s agar for 7 days. White arrows indicate terminal and intercalary phialides, while black arrows indicate the yeast-like cells

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For the molecular identification, genomic DNA was extracted using the MasterPure Yeast DNA Purification Kit (Epicentre Biotechnologies, USA) following the instructions of the manufacturer. PCR amplification was carried out by targeting the internal transcribed spacer (ITS) region using ITS1 and ITS4 primers [19]. The sequences were determined by LGC Genomics GmbH (Germany), and the consensus of the two reads was assembled by using the Staden package (Pregap4 v.1.5 and Gap4 v4.10) [20] and manually edited in BioEdit [21]. The obtained 592 bp long fragment was compared with the available sequences in the GenBank using the Basic Local Alignment Search Tool (BLAST) [22]. The isolate was finally identified as E. dermatitidis, as its ITS sequence displayed a 99% sequence homology with the ITS region of E. dermatitidis CBS 149.90 (Table 2). While the isolate itself was deposited in the Szeged Microbiological Collection (SZMC, Szeged, Hungary; http://szmc.hu/) under the strain number of SZMC 21989, its ITS sequence was deposited in the EMBL Nucleotide Sequence Database under the accession number of LN809939.

Table 2 Top five results of nucleotide–nucleotide BLAST search performed with the ITS sequence of the case isolate in the National Center for Biotechnology Information database (NCBI, http://www.ncbi.nlm.nih.gov/)
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The antifungal susceptibility profile of the case isolate was determined to 12 clinically relevant antifungal agents. The Etest method (BioMérieux, France) was used to determine the susceptibility to echinocandins (anidulafungin, caspofungin, micafungin), ketoconazole and posaconazole according to the instructions of the manufacturer. Minimum inhibitory concentration (MIC) values of the remaining seven antifungals were determined in accordance with the instructions of the CLSI (Clinical and Laboratory Standards Institute) M38-A2 broth microdilution method [23], with the involvement of the following commercially available standard powders and eye drops: amphotericin B, (Amphocare injection, BPRL Pvt ltd, Bangalore, India), clotrimazole (Auroclot, 1% (w/v) suspension, Aurolab, Madurai, India), econazole (Aurozole, 2% (w/v) suspension, Aurolab, Madurai, India), fluconazole (0.3% (w/v), Zocon, FDC ltd, Aurangabad, India), itraconazole (Itral, 1% (w/v) suspension, Jawa Pharmaceuticals, Guargon, India), natamycin (Natamet, 5% (w/v) suspension, Sun Pharmaceuticals Ind. Ltd., India) and voriconazole (Vfend, Pfizer Inc., India). Three replicates were involved in all the susceptibility tests. The obtained MIC values are summarized in Table 3. MIC was defined as the lowest antifungal concentration, which was required for the total growth inhibition of the test isolate. Susceptibility testing revealed that the MICs of amphotericin B, clotrimazole, econazole, fluconazole, itraconazole, ketoconazole, posaconazole and voriconazole were > 0.5 µg/ml, whereas MICs for natamycin and echinocandins were 8 µg/ml and > 32 µg/ml, respectively.

Table 3 Antifungal susceptibility profile of Exophiala dermatitidis isolate SZMC 21989
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Discussion and Conclusions

According to the available literature in the PubMed database (http://www.ncbi.nlm.nih.gov/pubmed), only three Exophiala species, E. jeanselmei, E. dermatitidis and E. phaeomuriformis have been identified from various human eye infections such as keratitis, endophthalmitis and subconjunctival mycetoma in the last decades (Table 1). Until now, six Exophiala endophthalmitis cases have been reported from different parts of the world. The first case of endophthalmitis caused by E. dermatitidis was described by Margo et al. in 1990 [14]. Since then, two other cases were reported by Benaoudia et al. in 1999 [15] and Clamp et al. in 2014 [17]. Medical therapy included surgical intervention supplemented with either intravitreal (2.5–5.0 µg) or intravenous (0.2–0.8 mg/kg) injections of amphotericin B in all cases. Benaoudia et al. [15] also used oral itraconazole (200 mg b.i.d.) and amphotericin B eye drops (0.15% (w/v) eight times a day). A failed therapeutic approach and consequent enucleation of the blind eyes were reported in the cases of Margo et al. [14] and Clamp et al. [17]. Endophthalmitis cases due to another species, E. jeanselmei, were also treated with systemic and intraocular amphotericin B, but ocular atrophy developed in both cases [16]. These five, above mentioned patients, suffered from different underlying disorders such as diabetes, Crohn’s disease and purpura. Based on these prior reports of Exophiala eye infections (Table 1), underlying conditions seem to increase the chances of a failed therapy. Infections were eradicated in almost all seven cases of keratitis. Conversely, subconjunctival mycetoma and endophthalmitis cases usually had poor visual outcome. In comparison to previous reports, our patient had no other complaints, and after vitrectomy he was successfully treated with intravitreal injections and eye drops of voriconazole and oral fluconazole. Based on the experience, authors recommend prompt removal of the intraocular lens and capsular bag along with vitrectomy to eradicate the infection. Also in eyes with localized growth (like in our case), care should be taken to reach the area of growth when taking specimens for culture and staining to increase the chance of getting a positive yield unlike in routine endophthalmitis cases where the specimen is usually taken from the center of the vitreous cavity. In the presented case, the surgeon took utmost care to get the repeat specimen from the suspected area of the exudate as the first specimen proved to be negative.

While in the 1990s mainly amphotericin B was chosen for the treatment of Exophiala eye infections, it was replaced in the last few years by new azole compounds, like voriconazole (Table 1). Unfortunately, limited data are available about the in vitro antifungal activity of these new agents against Exophiala isolates derived from human eye infections. Based on the in vitro antifungal susceptibility tests, the case isolate had variable susceptibility to different antifungal drugs (Table 3). Basically, the obtained results agreed with the previous report of Chowdhary et al. [5], except from the finding that echinocandins (i.e., anidulafungin, caspofungin, micafungin) proved to be ineffective against the tested isolate in the investigated concentration range (MIC > 32 µg/ml), while Chowdhary et al. [5] found a much lower MIC range (0.25–8 µg/ml) for E. dermatitidis strains.

In conclusion, endophthalmitis due to E. dermatitidis is a rare but serious infection of the eye. Its management requires a radical surgical intervention for a successful outcome, and also utmost care is necessary in collecting vitreous sample in eyes with localized infection to achieve higher culture positivity rate. The subsequent application of a carefully chosen, effective antifungal therapy is critical for a successful outcome. This case obviously demonstrates that besides Aspergillus [24], Curvularia [25] and Fusarium [26, 27] species, Exophiala dermatitidis should also be taken into consideration as a causative agent of eye infections in South India.