Case of Keratitis Caused by Aspergillus tamarii

Mycological study and diagnosis.The fungus was subcultured on malt extract agar plates and identified as Aspergillus tamarii Kita based on the colony morphology and microscopic features of the isolate (Fig. 1 and 2). Colonies on malt extract agar at room temperature attained diameters of 6.0 to 7.0 cm in 10 days, producing abundant conidial heads in dull yellowish green shades becoming metallic bronze at maturity (24). The conidiophore stipe was hyaline and rough walled; the conidial heads were radiate; the vesicles were globose to subglobose, 25 to 50 μm in diameter. The phialides were borne directly on the vesicle or on metulae (mostly on large heads). The conidia were globose to subglobose, 5 to 6.5 μm in diameter, and brownish yellow. However, in contrast with those of typical wild A. tamarii isolates (Fig. 2A), some conidia of this isolate were not ornamented with tubercules and warts but were smooth walled and hyaline (Fig. 2B). The isolate grew well at 37°C but was unable to grow at 42°C on malt extract agar medium.

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FIG. 1.

Conidial head of A. tamarii 2342/05.

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FIG. 2.

Conidia of a typical A. tamarii strain (CBS 484.65) (A) and that of A. tamarii 2342/05 (B). The scale bar represents 10 μm.

For purposes of molecular identification, mycelia grown in liquid YPG medium (0.5% Bacto yeast extract, 0.5% Bacto peptone, 1% glucose) for 1 day were subjected to DNA isolation by a Masterpure yeast DNA purification kit (Epicenter Biotechnologies, Madison, WI) according to the manufacturer's instructions. The internal transcribed spacer (ITS) region of the rRNA gene complex, incorporating ITS 1, the 5.8S rRNA gene, and ITS 2, was amplified using primers ITS1 and ITS4 (26). A segment of the calmodulin gene was amplified using primers cmd5 and cmd6 as described by Hong et al. (12), while a segment of the β-tubulin gene was amplified using primers bT2a and bT2b (9). DNA sequences were determined using a BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems Inc., Foster City, CA) and an ABI 3100 DNA sequencer. Both strands of each fragment were sequenced. The resulting sequences were deposited in the GenBank database. Sequence analysis was carried out by a BLASTN similarity search (2) at the website of the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/blast/ ).

Table 1 lists A. tamarii sequences with complete homology to those of the case isolate. The ITS, tubulin, and calmodulin sequences of the case isolate proved to be completely identical to the corresponding sequences of A. tamarii strain NRRL 25565 (13).

Living cultures were deposited at the Department of Microbiology, Aravind Eye Hospital and Postgraduate Institute of Ophthalmology, Coimbatore, India (strain number 2342/05), and at the Centraalbureau voor Schimmelcultures (CBS 121598).

Antifungal susceptibility testing.The Etest method (AB BIODISK, Solna, Sweden) for molds was applied for the determination of MICs of amphotericin B, fluconazole, itraconazole, ketoconazole, and voriconazole according to the manufacturer's instructions on RPMI 1640 agar (15 g in 1,000 ml) supplemented with 20 g glucose per 1,000 ml medium (1). The Etest drug concentrations ranged from 0.016 to 256 μg/ml for fluconazole and from 0.002 to 32 μg/ml for itraconazole, ketoconazole, voriconazole, and amphotericin B. The MIC was read as the drug concentration at which the elliptical inhibition zone intersected the scale of the Etest strip. The MICs of natamycin and econazole were determined by the broth microdilution method according to NCCLS M38-A in RPMI broth (18). The MICs for natamycin, amphotericin B, fluconazole, itraconazole, ketoconazole, voriconazole, and econazole proved to be >1,024, 0.125, >256, 0.064, 0.25, 0.125, and 0.064 μg/ml, respectively.

Discussion.Corneal infections of fungal etiology are very common and represent 30% to 40% of all cases of culture-positive infectious keratitis. Combating fungal keratitis is of special importance in India, which harbors the largest agrarian population at risk for developing blindness due to the limited availability of antifungal drugs and the lack of response during therapy. Certain Aspergillus species, mainly Aspergillus flavus (23, 25), Aspergillus terreus (25), Aspergillus fumigatus (23, 25), and Aspergillus niger (4), have long been regarded as important pathogens in eye infections, especially keratitis. Other members of the genus less frequently occurring in keratitis include Aspergillus glaucus and Aspergillus ochraceus. Most of the Aspergillus strains isolated from keratomycosis patients are being identified and reported at the genus level only (10). Their molecular identification at the species level would be of great importance, as the pathogenic potential may vary between different species of the genus.

A. tamarii is a member of Aspergillus section Flavi (8). This species is widely used in the food industry for the production of soy sauce (known as red Awamori koji) (14) and in the fermentation industry for the production of various enzymes, including amylases, proteases, and xylanolytic enzymes (3, 7, 17). Although A. tamarii is able to produce several toxic secondary metabolites, including cyclopiazonic acid and fumigaclavines (24), it has rarely been encountered as a human pathogen. The only known cases are an eyelid infection (5), invasive nasosinusal aspergillosis in an immunocompetent patient (20), and onychomycosis in a 3-year-old boy (15). To our knowledge, the present case of fungal keratitis is the first report on an ocular infection caused by A. tamarii and the fourth known case worldwide involving this unusual opportunistic human pathogen.

Nucleotide sequence accession numbers.The GenBank accession numbers for the sequences of the case isolate are EF525554 (ITS), EF525555 (β-tubulin), and EF525556 (calmodulin).