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Rapid detection and identification of Candida, Aspergillus, and Fusarium species in ocular samples using nested PCR
E E Jaeger1, N M Carroll, S Choudhury
1Department of Clinical Ophthalmology, The Institute of Ophthalmology and Moorfields Eye Hospital, London EC1V 9EL, United Kingdom.
Abstract:
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.
Insights
A new protocol rapidly detects fungal DNA in eye samples using panfungal and species-specific PCR. This method shows high sensitivity, potentially exceeding traditional culture for diagnosing fungal endophthalmitis.
Area of Science:
- Ophthalmology
- Molecular Biology
- Mycology
Background:
- Fungal endophthalmitis diagnosis can be challenging.
- Current diagnostic methods may lack sensitivity or speed.
Purpose of the Study:
- To develop a rapid and sensitive molecular protocol for detecting fungal DNA in ocular samples.
- To identify specific fungal species commonly causing endophthalmitis.
Main Methods:
- Designed novel panfungal primers targeting 18S rRNA for broad fungal detection.
- Employed a nested PCR approach with species-specific primers for Candida albicans, Aspergillus fumigatus, and Fusarium solani.
- Developed a streamlined DNA extraction protocol to minimize loss and remove PCR inhibitors from ocular fluids.
Main Results:
- Achieved high PCR sensitivity, detecting as little as 50-100 fg of free DNA and 1-2 Candida albicans organisms.
- Preliminary patient sample results correlated with clinical data.
- Identified one case positive by PCR but negative by culture, suggesting superior sensitivity.
Conclusions:
- The developed protocol offers rapid and sensitive detection of key fungal pathogens in ocular samples.
- This molecular system may improve the diagnosis and management of suspected fungal endophthalmitis.
- The protocol's sensitivity may surpass conventional culture techniques.