Rapid detection and identification of Candida, Aspergillus, and Fusarium species in ocular samples using nested PCR.
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.
Department of Clinical Ophthalmology, The Institute of Ophthalmology and Moorfields Eye Hospital, London EC1V 9EL, United Kingdom.
E E
EE
Jaeger
N M
NM
Carroll
S
S
Choudhury
A A
AA
Dunlop
H M
HM
Towler
M M
MM
Matheson
P
P
Adamson
N
N
Okhravi
S
S
Lightman
D001228:Aspergillosis / Q000175:diagnosis / Q000382:microbiology; D001232:Aspergillus fumigatus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D002176:Candida albicans / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D002177:Candidiasis / Q000175:diagnosis / Q000382:microbiology; D017931:DNA Primers; D004271:DNA, Fungal / Q000032:analysis; D009877:Endophthalmitis / Q000382:microbiology; D015821:Eye Infections, Fungal / Q000175:diagnosis / Q000382:microbiology*; D005670:Fusarium / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013045:Species Specificity; D014821:Vitrectomy; D014822:Vitreous Body / Q000382:microbiology
D017931:DNA Primers; D004271:DNA, Fungal