28732879
0095-1137/01/$04.000 DOI: 10.1128/JCM.39.8.28732879.2001
Copyright 2001, American Society for Microbiology. All Rights Reserved.
Standard fungal isolates. (i) Strains. Clinical and standard isolates of Aspergillus, Candida, Fusarium, Scedosporium, Alternaria, and Cryptococcus were used in
this study (Table 1). Strains were cultured on Sabouraud dextrose broth (2%
2873
The goal of this study was to determine whether sequence analysis of internal transcribed spacer/5.8S
ribosomal DNA (rDNA) can be used to detect fungal pathogens in patients with ocular infections (endophthalmitis and keratitis). Internal transcribed spacer 1 (ITS1) and ITS2 and 5.8S rDNA were amplified by PCR
and seminested PCR to detect fungal DNA. Fifty strains of 12 fungal species (yeasts and molds) were used to
test the selected primers and conditions of the PCR. PCR and seminested PCR of this region were carried out
to evaluate the sensitivity and specificity of the method. It proved possible to amplify the ITS2/5.8S region of
all the fungal strains by this PCR method. All negative controls (human and bacterial DNA) were PCR
negative. The sensitivity of the seminested PCR amplification reaction by DNA dilutions was 1 organism per
PCR, and the sensitivity by cell dilutions was fewer than 10 organisms per PCR. Intraocular sampling or
corneal scraping was undertaken for all patients with suspected infectious endophthalmitis or keratitis
(nonherpetic), respectively, between November 1999 and February 2001. PCRs were subsequently performed
with 11 ocular samples. The amplified DNA was sequenced, and aligned against sequences in GenBank at the
National Institutes of Health. The results were PCR positive for fungal primers for three corneal scrapings, one
aqueous sample, and one vitreous sample; one of them was negative by culture. Molecular fungal identification
was successful in all cases. Bacterial detection by PCR was positive for three aqueous samples and one vitreous
sample; one of these was negative by culture. Amplification of ITS2/5.8S rDNA and molecular typing shows
potential as a rapid technique for identifying fungi in ocular samples.
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FERRER ET AL.
J. CLIN. MICROBIOL.
Straina
ITS1ITS4
ITS86ITS4
570
595
596
599
608
536
520
524
820
510
544
569
292
300
299
300
308
282
255
270
360
294
283
286
556
556
611
320
320
329
a
IOA; ocular isolates obtained from the Instituto Oftalmolo
gico de Alicante,
Alicante, Spain; HMM, Ocular isolates obtained from Mo
stoles Hospital, Madrid, Spain; SCNC, Clinical isolates obtained from Spanish Cryptococcus neoformans Collection, Mycology Laboratory, Universidad Miguel Hernandez, Alicante, Spain; CECT, Spanish Type Culture Collection, Valencia, Spain; ATCC,
Americans Type Culture Collection. Rockville, Md.
RESULTS
Standard fungal isolates. (i) PCR specificity. The primers
used in this study (ITS1 and ITS4 for the first round amplification and ITS86 and ITS4 for the second round) successfully
amplified DNA from all the standard fungal strains tested.
After the first round of amplification, a product of approximately 550 bp was obtained. After the second round of amplification, the fragment obtained was about 280 bp (Fig. 1).
No amplification products were detected by using the ITS1-
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FERRER ET AL.
J. CLIN. MICROBIOL.
FIG. 4. (A) First PCR (ITS1-ITS4 primer pair) from different ocular samples. M, ladder marker GeneRuler 100bp DNA Ladder Plus
(500 bp, black triangle); (B) Seminested PCR product (ITS86-ITS4
primer pair). M, Ladder marker pBR322 DNA/BsuRI (434 bp, white
triangle; 267 bp, black triangle); P1, patient 1; P2, patient 2; P3, patient
3; P4, patient 4; P5Aq, patient 5 aqueous sample; P5Vit, patient 5
vitreous sample; P6, patient 6; P7Aq, patient 7 aqueous sample; P7Vit,
patient 7 vitreous sample.
2877
TABLE 2. Gram stain, culture, and PCR results with samples from 10 patients with a clinical diagnosis of endophthalmitis or keratitisa
Patient
Sample
Culture result
1
2
3
4
5
5
6
7
7
8
9
10
Corneal Scrape
Aqueous
Aqueous
Aqueous
Aqueous
Vitreous
Corneal scrape
Aqueous
Vitreous
Aqueous
Corneal scrape
Culture of corneal scrape
Positive
ND*
ND*
ND*
ND*
Positive
Negative
ND*
Negative
ND*
Negative
Positive
Positive (2 days)
Positive (3 days)
Positive (6 days)
Negative
Positive (13 days)
Positive (7 days)
Negative
Negative
Positive (7 days)
Negative
Positive (7 days)
Positive
ND
Positive/negative
Negative
Negative
Positive/positive
Positive/positive
ND
Negative
Negative
Positive/negative
ND
ND
Positive/A. fumigatus
Negative
Positive/C. parapsilosis
Negative
Negative
Negative
Positive/A. niger
Negative
Positive/C. parapsilosis
Negative
Positive/Alternaria alternata
Positive/S. apiospermum
a
b
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FERRER ET AL.
ACKNOWLEDGMENTS
This work was supported by grant IMTEIA/1998/210 from the
IMPIVA (Generalitat Valenciana, Spain) and a grant from Instituto
Oftalmolo
gico de Alicante (Alicante, Spain).
We thank Gema Salas, Stuart Ingham, and Maria Luz Campos
(Facultad de Medicina, Universidad Miguel Hernandez, Alicante, Spain)
for their technical assistance; Kathy Hernandez for her English language corrections; and Josefa Anto
n for scientific suggestions. We also
thank Elisa Amor from Mo
stoles Hospital (Madrid, Spain) for her collaboration with the study of the Scedosporium apiospermum strain and
for providing information on the keratitis case associated with this strain.
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