title: Molecular characterization of drug-resistant and drug-sensitive Aspergillus isolates causing infectious keratitis.
abstract: OBJECTIVE
  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.
  
  
  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).
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Ocular Microbiology, Dr. Rajendra Prasad Centre for Ophthalmic Science, New Delhi-110 029, India. niruni2000@yahoo.com
authors.forename: Niranjan
authors.initials: N
authors.lastname: Nayak
authors.forename: Gita
authors.initials: G
authors.lastname: Satpathy
authors.forename: Sujata
authors.initials: S
authors.lastname: Prasad
authors.forename: Jeewan S
authors.initials: JS
authors.lastname: Titiyal
authors.forename: R M
authors.initials: RM
authors.lastname: Pandey
authors.forename: Rasik B
authors.initials: RB
authors.lastname: Vajpayee
mesh_terms: D000935:Antifungal Agents / Q000494:pharmacology*; D001228:Aspergillosis / Q000188:drug therapy / Q000382:microbiology*; D001230:Aspergillus / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification*; D003315:Cornea / Q000382:microbiology*; D025141:Drug Resistance, Fungal; D015821:Eye Infections, Fungal / Q000175:diagnosis / Q000382:microbiology*; D006801:Humans; D007634:Keratitis / Q000175:diagnosis / Q000382:microbiology*; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D012331:RNA, Fungal / Q000032:analysis
chemical_list: D000935:Antifungal Agents; D012331:RNA, Fungal