Keratitis due to Aspergillus flavus: Clinical profile, molecular identification of fungal strains and detection of aflatoxin production

Abstract

To document the clinical profile of patients with keratitis due to Aspergillus flavus and to elaborate on differences in the aflatoxin-producing potential of keratitis strains versus environmental strains of A. flavus. Over a 6-month period, strains of Aspergillus flavus were isolated in culture from corneal scrape or biopsy material of patients who presented with suppurative keratitis (clinical isolates). The strains were confirmed to be A. flavus by molecular methods (amplification of the internal transcribed spacer 2 [ITS 2] region and direct sequencing followed by comparative GenBank analysis). The aflatoxin-producing potential of each strain was determined by thin-layer chromatography. The ability of each strain to form sclerotia in Czapek-Dox agar (CDA) after 7 days incubation at 30 °C in the dark and to produce a beige ring in yeast extract sucrose agar supplemented with methyl β-cyclodextrin and sodium desoxycholate (YESD medium) after 3 days incubation at 30 °C was also assessed. For comparison, the tests were also run on 10 strains of A. flavus (identity confirmed by molecular methods) collected from local farming areas (environmental isolates). Aflatoxin B1 was detected in 16 (80%) of 20 culture filtrate or mycelial homogenate samples of the clinical isolates (mean concentration: 366.7±125.4 parts per billion [ppb]) but in only eight (40%) of 20 samples of environmental isolates (mean concentration: 306.6±125.4 ppb). Seven of the eight aflatoxin-producing clinical isolates and two of the four aflatoxin-producing environmental isolates formed sclerotia (>400 μm) and a beige ring in culture. Aflatoxin B1 was detected in a significantly higher percentage of growth samples of clinical isolates (80%) than growth samples of environmental isolates (40%) (χ2=6.667; p=0.0098); the therapeutic implications of this finding require further study. The production of sclerotia and a beige ring in culture appear to be useful markers of aflatoxin-producing potential in strains of A. flavus isolated from keratitis.