Polymerase Chain Reaction Based Detection of Fungi in Suspected Infectious Keratitis Patients

Background & Objectives: Fungal infections of the eye are rampant in tropical regions and are responsible for significant ocular morbidity and blindness. Prompt and specific identification of fungi is important in order to define clinical treatment. However, in most cases conventional culture identification can be considered to be time consuming, of low sensitivity and not without errors. The aim of the study is to evaluate a polymerase chain reaction (PCR) based assay for fungal infections in the eye. Methods: Polymerase chain reaction (PCR) for detection of fungal DNA was done with panfungal primers (ITS1 and ITS4) for diagnosis of fungal aetiology. Ocular samples (conjunctival swabs, corneal scrapings, corneal biopsies, vitreous aspirates and globe contents) from 30 patients with presumed fungal infection were evaluated using this assay, as well as by standard microbiological techniques, and the results were compared. Results: Thirty clinical specimens were evaluated by PCR and by other conventional techniques (culture and staining). Of the 30 specimens analyzed, fungal keratitis was definitively diagnosed by culture in 18 (60%). 17 (57%) of these 18 specimens were PCR positive. One specimen (3% of the 30 total) was fungal culture positive but PCR negative. 12 (40%) of 30 specimens were fungal culture negative, and 7 (23%) of these 12 were also PCR negative. Five (17%) were PCR positive but fungal culture negative. Of the seven specimens negative by both PCR and fungal culture, all showed no growth. All 30 specimens were also examined by light microscopy with potassium hydroxide (KOH) and Gram’s staining. KOH and/or Gram’s staining was found to be positive in 8 (27%) out of 30 cases. All positive smears revealed septate hyphae. 3 (38%) of these 8 specimens showed fungi on smear but were fungal culture negative, and two of these three specimens were PCR positive which were clinically to have fungal keratitis. Conclusions: PCR not only proved to be an effective rapid method for the diagnosis of fungal infections in eye but was also more sensitive than staining and culture methods. Due to promising results of the DNA extraction protocol and PCR assay in our laboratory, we were able to make early diagnosis. Hence, effective therapy was given, which had a major impact on the improvement in the prognosis of subject with fungal infections. Key-wordsFungal keratitis, Lyticase, Proteinase K, Polymerase chain reaction, PCR assay, DNA extraction -------------------------------------------------IJLSSR----------------------------------------------INTRODUCTION Fungal infections of the eye are on rise and are responsible for ocular morbidity and blindness. Access this article online