Investıgatıon of fungal agents on corneal samples by classıcal and molecular methods

Introduction and Aim: Corneal diseases are the second major cause of blindness other than cataracts. Among corneal diseases, fungal cataracts have been more frequently encountered for the last 20-30 years. Advances in diagnostic procedures and increased usage of corticosteroids or immun suppressive agents are thought to be responsible for this condition. Early diagnosis and early initiation of proper antifungal treatment are very important for the recovery of the disease. The aim of this study was to detect fungal agents and fungal particules by microscopic evaluation, to grow fungal agents by classical conventional methods and to identify fungal agents directly by polimerase chain reaction (PCR), from the samples obtained with the suspicion of fungal keratitis and evaluated in Micology Laboratory of Ege University School of Medicine Department of Medical Microbiology in one year period. In addition to conventional methods, routine application of the PCR method for searching fungus in micology labarotory practice was the secondary aim of the study. Methods: In this study, corneal samples were obtained by ophtalmologists, from the patients with the suspicion of fungal keratitis. The samples were used in three ways; for culture inoculation, for direct or stained microscopic evaluations and for molecular methods. Conjunctival swabs collected from eyes were inoculated in the shape of halfmoon onto sheep blood agar (bioMérieux, France), chocolate agar (bioMérieux, France), Eosin methylene blue agar (bioMérieux, France), Sabouraud dextrose agar (SDA) with antibiotic (bioMérieux, France) and Staib agar (in house) and all were incubated for 10 days. Microscopic evaluation was made by potassium hydroxide (KOH) 10% and Gram staining, in the Micology Laboratory of Ege University School of Medicine Department of Medical Microbiology. In case of detecting fungal agents on direct microscopic evaluations, clinicians were informed emergantly. Fungal agents growing in the culture were described by conventional methods and Vitek-MS (bioMérieux, France) automized system. Lastly, conventional PCR method was applied to the samples. Results: In a one year period, corneal scraping specimens were obtained from 60 patients, 37 (61,6%) of them were male and 23 (38,4%) were female. The median age of the patients was 49 (3-85). In direct microscopic evaluation of 5 (8,3%) of the 60 patients fungal hyphae were found. Growing in the culture was detected on 12 (20%) of the 60 samples. Seven (11,6%) of these growings on sheep blood agar (35°C) were considered as bacteria. On the five (8,3%) samples, growings in the SDA (26°C) with antibiotics were considered as fungal agents (yeasts and molds). By the PCR with parafungal primers of 60 samples, fungal deoxyribonucleic acid (DNA) were detected in the seven (11,6%) of them. In three of the five samples with fungal hyphae detected by direct evaluation, fungal agents were detected both by culture and conventional PCR method; in the rest of two samples fungal agents did not grow in culture but fungal DNA of them were detected by conventional PCR method. In two of the samples, in which fungal agents grew in the culture and fungal DNA's were be detected by conventional PCR, fungal hyphae could not be demonstrated by direct microscopic evaluation. Conclusion: In this study, fungal keratitis were demonstrated in nearly 12% of the patients. The results of direct microscopic evaluation, culture and conventional PCR were found compatible. Demonstration of fungal existance by conventional PCR method in corneal samples, caused determination of fungal agents which did not grow in the culture and moreover shortened the time passed for diagnosis in comparison with the classical conventional methods. The usage of this method in clinical microbiology practice will protect from eye loss by early initiaion of antimicotic treatment. Key words: Keratitis; Fungal keratitis; Direct microscopic evaluation; KOH; Culture; Conventional PCR.