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Journal of Clinical Microbiology, August 2000, p. 2902-2908, Vol. 38, No. 8
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Rapid Detection and Identification of Candida, Aspergillus, and Fusarium Species in Ocular Samples Using Nested PCR

Emma E. M. Jaeger,1,dagger Nora M. Carroll,1,Dagger Sarah Choudhury,1 Anthony A. S. Dunlop,1 Hamish M. A. Towler,2 Melville M. Matheson,3 Peter Adamson,1 Narciss Okhravi,1,* and Susan Lightman1

Department of Clinical Ophthalmology1 and Department of Pathology,3 The Institute of Ophthalmology and Moorfields Eye Hospital, London EC1V 9EL, and Department of Opthalmology, Whippscross Hospital, London E11 1NR,2 United Kingdom

Received 13 January 2000/Returned for modification 8 May 2000/Accepted 3 June 2000

A protocol for the rapid detection of fungal DNA in ocular samples, derived from three species, Candida albicans, Aspergillus fumigatus, and Fusarium solani, has been developed. Two novel panfungal primers complementary to 18S rRNA sequences present in all three species were designed. Panfungal PCR was followed by three nested PCRs utilizing species-specific primers. PCR sensitivity ranged from 50 to 100 fg of free DNA and between one and two C. albicans organisms. In addition, we also developed a rapid and reliable DNA extraction protocol. This protocol minimized DNA loss during extraction, whilst removing compounds from vitreous and aqueous fluids that have previously been shown to have inhibitory effects on PCR. Preliminary results obtained after testing the protocol on three patient samples support culture results and medical history. However, one patient was PCR positive but culture negative, suggesting that the sensitivity of this protocol may exceed that of traditional culture techniques. This system, therefore, constitutes an additional protocol that may significantly aid patient management in cases where fungal endophthalmitis is suspected.

* Corresponding author. Mailing address: Department of Clinical Ophthalmology, The Institute of Ophthalmology and Moorfields Eye Hospital, 11-43 Bath St., London EC1V 9EL, United Kingdom. Phone and Fax: 44-(0)171-608-6931. E-mail: nokhravi{at}hgmp.mrc.ac.uk.

dagger Present address: Molecular and Population Genetics Laboratory, Imperial Cancer Research Fund, London WC2A 3PX, United Kingdom.

Dagger Present address: Department of Medical Biochemistry, University of Stellenbosch, Tygerberg 7505, South Africa.

Journal of Clinical Microbiology, August 2000, p. 2902-2908, Vol. 38, No. 8
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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