Molecular characterization of drug-resistant and drug-sensitive Aspergillus isolates causing infectious keratitis

PURPOSE: To study the susceptibilities of Aspergillus species against amphotericin B in infectious keratitis and to find out if drug resistance had any association with the molecular characteristics of the fungi. MATERIALS AND METHODS: One hundred and sixty Aspergillus isolates from the corneal scrapings of patients with keratitis were tested for susceptibilities to amphotericin B by broth microdilution method. These included Aspergillus flavus (64 isolates), A. fumigatus (43) and A. niger (53). Fungal DNA was extracted by glass bead vertexing technique. Polymerase chain reaction (PCR) assay was standardized and used to amplify the 28S rRNA gene. Single-stranded conformational polymorphism (SSCP) of the PCR product was performed by the standard protocol. RESULTS: Of the 160 isolates, 84 (52.5%) showed low minimum inhibitory concentration (MIC) values (≤ 1.56 μg/ml) and were designated as amphotercin B-sensitive. Similarly, 76 (47.5%) had high MICs (≥ 3.12 μg/ml) and were categorized as amphotericin B-resistant. MIC(50) and MIC(90) values ranged between 3.12-6.25 μg/ml and 3.12-12.5 μg/ml respectively. A. flavus and A. niger showed higher MIC(50) and MIC(90) values than A. fumigatus. The SSCP pattern exhibited three extra bands (150 bp, 200 bp and 250 bp each) in addition to the 260 bp amplicon. Strains (lanes 1 and 7) lacking the 150 bp band showed low MIC values (≤ 1.56 μg/ml). CONCLUSION: A. niger and A. flavus isolates had higher MICs compared to A. fumigatus, suggesting a high index of suspicion for amphotericin B resistance. PCR-SSCP was a good molecular tool to characterize Aspergillus phenotypes in fungal keratitis.