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

Abstract

Purpose: 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.

Methods: 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 degrees C in the dark and to produce a beige ring in yeast extract sucrose agar supplemented with methyl beta-cyclodextrin and sodium desoxycholate (YESD medium) after 3 days incubation at 30 degrees 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).

Results: 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 microm) and a beige ring in culture.

Conclusions: Aflatoxin B1 was detected in a significantly higher percentage of growth samples of clinical isolates (80%) than growth samples of environmental isolates (40%) (chi(2)=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.

Figure 1

Photomicrograph showing a septate, branching hypha (arrow) of Aspergillus flavus in corneal scrape material from a patient with keratitis (Gram stain; 1,000×). Direct microscopic examination of corneal material by the method of Gram staining revealed the presence of fungal hyphae.

Figure 2

Gel picture showing the PCR amplified product (350 bp) of the three isolates of Aspergillus flavus. Internal transcribed spacer 2 was used as a target region for DNA amplification. The lane L1 was loaded with 100 bp DNA Ladder; the lanes L2, L3, L4, L5 were loaded with 10 μl of the amplified product from the test strains C1, C2, E1, E2, respectively, in 1.5% agarose gels. Lane L6 was loaded with a negative control to rule out false-positive results.

Figure 3

Thin layer chromatograms of culture filtrates of two clinical and one environmental isolate of Aspergillus flavus. L1: aflatoxin B1 was not detected in the extract from the culture filatrate of test strain C2 (aflatoxin-non producing clinical strain). L2: aflatoxin B1 was detected in the extract from the culture filtrate of test strain C1 (aflatoxin-producing clinical strain). L3: aflatoxin B1 was detected in the extract from the culture filtrate of the test strain E1 (aflatoxin-producing environmental strain). L4: aflatoxin B1 standard was loaded for confirmation.

Figure 4

Seven day-old growth of a clinical isolate of Aspergillus flavus on Czapek-Dox agar incubated at 30 °C. A: Formation of sclerotia is noted in the culture (arrows in inset). Formation of sclerotia was noted significantly more frequently in aflatoxin-producing strains than in aflatoxin-nonproducing strains. B: Seven day-old growth of another clinical isolate of Aspergillus flavus on Czapek-Dox agar incubated 30 °C. This isolate has not formed sclerotia in culture.

Figure 5

Three day-old growth of the isolate of Aspergillus flavus on yeast extract sucrose agar (supplemented with methyl β-cyclodextrin and sodium desoxycholate), incubated at 30 °C. A: Three day-old growth of a clinical isolate of Aspergillus flavus on yeast extract sucrose agar (supplemented with methyl β-cyclodextrin and sodium desoxycholate), incubated at 30 °C. A beige ring (arrow) has formed around the colony. Formation of a beige ring was noted significantly more frequently in aflatoxin-producing strains than in aflatoxin-nonproducing strains. B: Three day-old growth of an environmental isolate of Aspergillus flavus on yeast extract sucrose agar supplemented with methyl β-cyclodextrin and sodium desoxycholate), incubated at 30 °C. There is no beige ring around the colony growth.