Rapid detection and identification of Candida, Aspergillus, and Fusarium species in ocular samples using nested PCR

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.

FIG. 1

Alignments of primer sequences and corresponding sequences from C. albicans (Caab), A. fumigatus (Asfu), and F. solani (Fuso). Residues that differ from the primer sequence at the top of each alignment are boxed.

FIG. 2

Results obtained after PCR with (A) Pffor-Pfrev2 and PCR with 1 μl of this with (B) Cafor2-Carev3, (C) Asfufor-Asfurev, and (D) Fusofor-Fusorev. DNA (1 ng) from the following species was used: C. albicans, A. fumigatus, and F. solani. Lane 4 contains no DNA.

FIG. 3

Results obtained after PCR with (A) Pffor-Pfrev2 and PCR with 1 μl of this with (B) Cafor2-Carev3. Ten-nanogram samples of DNA from the following species was used: (lanes 1 and 2) C. guilliermondii, (lanes 3 and 4) C. glabrata, (lanes 5 and 6) C. pelliculosa, (lanes 7 and 8) C. tropicalis, (lanes 9 and 10) C. parapsilosis, (lanes 11 and 12) C. krusei, (lane 13) no DNA.

FIG. 4

PCR of serially diluted DNA from C. albicans using (A) Pffor-Pfrev2 and PCR with 1 μl of this with (B) Cafor2-Carev3.

FIG. 5

PCR of DNA extracted from serially diluted C. albicans using (A) Pffor-Pfrev2 and PCR with 1 μl of this with (B) Cafor2-Carev3. DNA used was equivalent to the following average number of organisms: (lane 1) >500, (lane 2) 7.7, (lane 3) 3.3, (lane 4) 0.6. Controls were blank (lanes 5 and 6), and 10 pg of DNA derived from C. albicans (lane 7), A. fumigatus (lane 8) and F. solani (lane 9).

FIG. 6

PCR of eluate derived from vitreous of patient 1 using (A) Pffor-Pfrev2 and PCR with 1 μl of this with (B) Cafor2-Carev3, (C) Asfufor-Asfurev, and (D) Fusofor-Fusorev. Lane contents are as follows: (lanes 1 and 2) 20 μl of eluate from patient 1, (lanes 3 to 5) negative control, (lane 6) 10 pg of C. albicans DNA, (lane 7) 10 pg of A. fumigatus DNA, (lane 8) 10 pg of F. solani DNA, and (lane 9) no DNA.