Polymerase Chain Reaction (PCR) guided Diagnosis of Mycotic Keratitis: a Prospective Evaluation of its Efficacy and Limitations

¹ OPHTHALMOLOGY, ALL INDIA INSTITUTE OF MEDICAL SCIENCES, DR R P CENTRE, NEW DELHI, 110029, India
² OPHTHALMOLOGY, Dr.Rajendra Prasad Centre for Ophthalmic Sciences,All India Institute of Medical Sciences, New Delhi, India
³ OCULAR MICROBIOLOGY, Dr.Rajendra Prasad Centre for Ophthalmic Sciences,All India Institute of Medical Sciences, New Delhi, India

^* To whom correspondence should be addressed. E-mail: sujithnayanar{at}gmail.com.

	Abstract

PURPOSE: To assess the utility of Polymerase Chain Reaction (PCR) in diagnosing fungal keratitis, and compare its sensitivity and specificity with those of the conventional microbiological techniques in our laboratory. METHODS: A prospective non-randomized investigation was undertaken at our tertiary-care ophthalmic facility to evaluate 40 eyes of 40 patients with presumed fungal keratitis, both fresh and treated. Besides routine bacterial culture and sensitivity, corneal scrapings were also evaluated by fungal culture, smear with potassium hydroxide (KOH) and Gram's stains, and PCR. The conventional PCR technique (Lee & Taylor protocol, 1990) was followed with minor modifications to suit our set-up, using primers targeted to 28S rRNA sequence, which is common to all fungi causing corneal infections in tropical climes. RESULTS: Of the 40 presumed cases of mycotic keratitis (30 untreated), PCR showed positivity in 50%, culture in 25%, and hyphae showing in KOH and Gram's in 40% and 35% respectively. The sensitivities of PCR, KOH and Gram's were 70%, 60% and 40% respectively; their specificities being 56.7%, 66.7% and 66.7%. Amongst 10/40 eyes pretreated with antifungal agents, PCR was positive in 50%, but culture in 30%. The time taken for PCR assay was 4-8hr, while positive fungal cultures took at least 5-7days. CONCLUSIONS: PCR not only proved an effective rapid method for the diagnosis of fungal keratitis, but was also more sensitive in our hands than KOH wet mount and Gram's smear. Barring its potential limitations discussed, PCR remains a promising tool for faster diagnosis of fungal keratitis.

Key Words: corneal ulceration, MYCOTIC KERATITIS, PCR, KOH SMEAR, FUNGAL CULTURE, GRAMS STAINING

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