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JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 2001, p.

28732879
0095-1137/01/$04.000 DOI: 10.1128/JCM.39.8.28732879.2001
Copyright 2001, American Society for Microbiology. All Rights Reserved.

Vol. 39, No. 8

Detection and Identification of Fungal Pathogens by PCR and by ITS2


and 5.8S Ribosomal DNA Typing in Ocular Infections
S,2 EMILIA MULET,3
CONSUELO FERRER,1* FRANCISCA COLOM,2 SUSANA FRASE
L. ABAD,1 AND JORGE L. ALIO
1,3
JOSE
Departamento de Biologa Molecular, Instituto Oftalmolo
gico de Alicante, 03015 Alicante,1 and Div. Microbiologa,2
and Patologa y Ciruga-Div. Oftalmologa,3 Universidad Miguel Herna
ndez, 03550 Alicante, Spain
Received 14 March 2001/Returned for modification 4 April 2001/Accepted 3 June 2001

The microbiological spectrum of infectious endophthalmitis


shows that the percentage of isolates that are fungi is 8 to
18.5% (2, 7, 12, 22, 23) and in keratitis the rate is 16 to 35.9%
(8, 42). Clinical diagnosis of these ocular infections is confirmed by obtaining intraocular (aqueous or vitreous) specimens or corneal scrapings. However, standard microbiological
tests are positive in only 54 to 69% of endophthalmitis cases
(13, 22, 23) (by culture) and 80% (8) of keratitis cases (by
Gram and Giemsa stains and culture). In fungal infections,
even when positive, results usually take longer than a week
because these organisms are difficult to identify and/or are
slow-growing. Early diagnosis and rapid intervention is a critical element for an effective treatment of ocular infections.
This has led to the development of culture-independent diagnostic tests such as PCR. PCR-based detection methods with
universal primers for bacterial DNA in ocular samples (5, 16,
20, 21, 26, 27, 34, 36, 40) have recently been developed. For
detection of fungal pathogens, multicopy gene targets have
been evaluated for increasing the sensitivity (33, 39) and universal fungal PCR primers have been developed for broadening the range of detectable fungi (9, 14, 18, 31, 37). Studies on
fungal DNA detection in ocular samples have been performed
(3, 15, 17, 35); the small number of conidia in the samples, the
difficulty of DNA extraction (25, 43) (some filamentous fungi
have a sturdy cell wall which is resistant to standard DNA

extraction procedures for yeast and bacteria), and the presence


of PCR inhibitors in human specimens (45) are some of the
difficulties with fungal detection in ocular samples. The ideal
marker to detect a fungal infection should be present in all
fungal genera (but should contain enough internal variation in
its sequence to define a given species) and should be a multicopy gene to maximize the sensitivity of the detection method.
The rRNA genes are good candidates, since they are present in
high copy number and the sensitivity of their detection may be
dramatically increased by the use of nested PCR. The transcriptional unit is composed of 18S, 5.8S, and 28S rRNA genes.
Between the 18S and 5.8S and between the 5.8S and 28S
ribosomal DNA (rDNA) gene subunits are intergenic transcribed spacer regions (ITS1 and ITS2) that are not translated
into rRNA. Although rRNA genes are highly conserved the
ITS regions are divergent and distinctive (1, 6, 10, 29, 30, 41,
46). This report describes the application of molecular techniques (sequence analysis of PCR-amplified ITS2/5.8S rDNA)
for fungal detection in two sets of samples: serial dilutions of
different fungal strains and clinical samples obtained from patients with delayed postoperative endophthalmitis or keratitis.
The aim of this technique is to reduce the time required for
mycological diagnosis, increase the number of ocular samples
from which a confirmed diagnosis is made, and identify the
causative fungal agent.

* Corresponding author. Mailing address: Dpto. Biologa Molecular, Instituto Oftalmolo


gico de Alicante, Avenida de Denia no. 111,
03015 Alicante, Spain. Phone: 34 965 154062. Fax: 34 965 160468.
E-mail: cferre@umh.es.

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%

MATERIALS AND METHODS

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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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J. CLIN. MICROBIOL.

TABLE 1. Strains and source of ocular isolates analyzed by PCR


amplification of rDNA

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.

[wt/vol] glucose, 1% [wt/vol] peptone) supplemented with chloramphenicol (1


mg liter1), subcultured onto Sabouraud dextrose agar slants, and kept at 4C.
(ii) Fungal DNA extraction. DNA extraction, preparation of the PCR mixture,
and post-PCR analysis were carried out in separate rooms using equipment
designated for each area to minimize the possibility of specimen contamination.
The type strains (Table 1) were inoculated in 1.5-ml Eppendorf tubes containing 0.5 ml of Sabouraud dextrose broth supplemented with chloramphenicol
and incubated overnight in an orbital shaker at 150 rpm and 30C. Thereafter,
fungal cultures were adjusted photometrically (absorbance at 530 nm; McFarland 0.5 standard) to a concentration of 1 106 to 5 106 cells/ml. In the case
of filamentous fungi, conidia were separated from the rest of the mycelium by
filtration through sterile glass wool (28). Tenfold serial dilutions of Candida
albicans and Aspergillus fumigatus (106 to 100 cells) were prepared to test the
sensitivity and specificity of the assay. The fungal suspensions with predetermined concentrations were centrifuged at 5,000 g, and then the pellet was
frozen at 20C for 1 h and incubated at 65C for 1 h in 0.5 ml of extraction
buffer (50 mM Tris-HCl, 50 mM EDTA, 3% sodium dodecyl sulfate, 1% 2-mercaptoethanol). The lysate was extracted with phenol-chloroform-isoamyl alcohol
(25:24:1, vol/vol/vol). Then, 65 l of 3 M sodium acetate and 75 l of 1 M NaCl
were added to 350 l of the supernatant and the resulting volume was incubated
at 4C for 30 min. DNA was recovered by isopropanol precipitation and washed
with 70% (vol/vol) ethanol. The concentration was measured by monitoring the
UV absorbance at 260 nm (Gene Quant System; Pharmacia, LKB Biochrom).
Serial aqueous dilutions of DNA from C. albicans and A. fumigatus were prepared to different concentrations (10 ng to 1 fg per 10 l) and stored at 20C.
(iii) Negative controls. (a) Extraction of DNA from human leukocytes. Human
DNA from whole blood was isolated by using the InstaGene Matrix (Bio-Rad
Laboratories, Hercules, Calif.) as specified by the manufacturer.
(b) Extraction from bacteria. A variety of bacterial organisms capable of
producing ocular infections were used to determine the specificity of the fungal
primers: Staphylococcus epidermidis, Pseudomonas aeruginosa, Escherichia coli,
and Streptococcus pneumoniae from the Spanish Type Culture Collection. Bacterial DNA was isolated by using the InstaGene Matrix.
(iv) PCR assay. Extracted DNA was amplified using a RoboCycler 96 temperature cycles (Stratagene, La Jolla, Calif). The primers and PCR conditions
used are specified below. PCR amplification was carried out in two steps.
(a) First-round amplification. The universal primers used for fungal amplification were ITS1 (5TCC GTA GGT GAA CCT GCG G 3), which hybridizes
at the end of 18S rDNA, and ITS4 (5TCC TCC GCT TAT TGA TAT GC 3),
which hybridizes at the beginning of 28S rDNA (44) (Life Technologies, Barcelona, Spain). The 50-l PCR mixture contained 10 l of DNA template, 6 l of

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Alternaria alternata IOA-K3


Aspergillus flavus CECT 2684
Aspergillus fumigatus CECT 2071/IOA-K1
Aspergillus niger CECT 2574/IOA-K2
Aspergillus terreus CECT 2748
Candida albicans CECT 1472
Candida parapsilosis ATCC 22019/IOA-E1, IOA-E2
Candida tropicalis CECT 1005
Candida glabrata CECT 1448
Candida kruseii ATCC 6258
Fusarium oxysporum CECT 2154
Fusarium solani CECT 2199
Cryptococcus neoformans
var. neoformans SCNC SP1
var. gatti SCNC C48
Scedosporium apiospermum HMM-K1

Predicted size (bp)


of PCR product
with primers:

25 mM MgCl2, 5 l of PCR buffer without MgCl2; 200 M each deoxynucleoside


triphosphate, 25 pmol of each primer, and 1 U of Taq DNA polymerase (Biotools
B&M Labs, S.A., Madrid, Spain). Reactions involved 1 cycle at 95C for 5 min,
followed by 35 cycles with a denaturation step at 95C for 30 s, an annealing step
at 55C for 1 min, and an extension step at 72C for 1 min, followed by 1 cycle
at 72C for 6 mins.
(b) Seminested amplification. For the second amplification, the primers used
were ITS86 (5GTG AAT CAT CGA ATC TTT GAA C 3), which hybridizes
with the 5.8S rDNA region (29), and ITS4 (Life Technologies, Barcelona, Spain).
Seminested PCR amplification mixtures contained 1 1 of first-round product in
50 l of PCR reaction mixture (6 l of 25 mM MgCl2, 5 l of PCR buffer without
MgCl2, 200 M each deoxynucleoside triphosphate, 50 pmol of primer ITS4, and
100 pmol of primer ITS86, and 1 U of Taq DNA polymerase (Biotools B&M
Labs). Reactions involved 1 cycle at 95C for 5 min, followed by 30 cycles with a
denaturation step at 95C for 30 s, an annealing step at 55C for 30 s, and an
extension step at 72C for 30 s, followed by 1 cycle at 72C for 6 min.
(c) Negative controls. Two negative controls were included in the first amplification: a reagent control (sterile water) and a sample extraction control. The
sample extraction control consisted of sterile MilliQ water subjected to the same
extraction procedures as the specimens. In the seminested PCR, 1 l each of the
two negative control samples from the first-round amplification and a third
negative control of sterile water were included.
(v) Detection of the amplified products. Aliquots (10 l) of each amplified
product were electrophoretically separated in a 2% agarose gel in 1 Trisborate-EDTA buffer and visualized using ethidium bromide under UV illumination. Molecular weight ladders were included in each run (pBR322 DNA/
BsuRI or Gene Ruler 100-bp DNA Ladder Plus [MBI Fermentas, Vilnius,
Lithuania]).
Ocular samples. (i) Patient selection. Intraocular sampling or corneal scrapings were undertaken for all patients with suspected infectious endophthalmitis
or keratitis (nonherpetic), respectively, between November 1999 and February
2001. Before sampling, informed consent was obtained from all patients. The
protocol for collection of aqueous samples, vitreous samples, and corneal scrapings was approved by the Institutional Review Board at the Instituto Oftalmolo
gico de Alicante, Alicante, Spain. This research followed the tenets of the
Declaration of Helsinki at all times.
(ii) Sample collection and culture. (a) Procedure for endophthalmitis cases.
The extraocular environment was sterilized with 5% povidone iodine solution
before surgery. Approximately 100 to 200 l of aqueous fluid was withdrawn
using a 30-gauge needle with a limbal paracentesis. Vitreous samples (200 l)
were taken at the time of three-port pars plana vitrectomy. The samples were
divided into two aliquots and transported to the microbiology laboratory and to
the molecular biology laboratory at 4C. One portion was immediately examined
by conventional microbiological diagnostic tests, and the other was frozen at
20C until processed by PCR.
For the microbiological diagnostic test, 50 l of aqueous humor or 50 l of
vitreous was cultured at 30C in Sabourauds dextrose agar or at 37C in thioglycolate broth, blood agar, chocolate agar, CLED agar, or MacConkey agar.
Bacteria were identified by the API Staph and API 20A systems (Biomeriux,
bioMerieux Sa, Marcy L Etoile, France). Yeasts were identified by the Auxacolor
system (Sanofi Diagnostics Pasteur, Inc, Marnes-la-Coquette, France), and filamentous fungi were differentiated by isolation in Sabouraud dextrose agar plus
chloramphenicol and morphological examination of a macroscopic and microscopic characteristics.
(b) Procedure for keratitis cases. Upon completion of the ocular examination
and after instillation of topical anesthetic, a sterile Kimura spatula was used to
scrape the area of infection. Scrapings were inoculated into thioglycolate broth,
Roiron broth, and Lo
wenstein-Jensen medium and were placed onto glass slides
for staining with Gram and Giemsa stains. The PCR sample was obtained by
scraping and stirring the spatula for a few seconds in 100 l of sterile water in a
1.5-ml sterile Eppendorf tube. Two aliquots of 50 l were taken from each
sample and stored at 20C.
(iii) Fungal DNA extraction. A 50-l volume of each ocular sample was frozen
for at least 1 h at 20C. The DNA extraction was performed as described for
the standard fungi isolates. DNA was diluted in 10 l of sterile water.
(iv) PCR assay. The PCR for fungal DNA detection was performed as described for the standard fungal isolates. The bacterial PCR and specific Propionibacterium acnes PCR amplification with ocular samples were performed as
described by Hykin et al. (16).
(a) Detection of PCR inhibitors in ocular samples. The presence of PCR
inhibitors in ocular fluids was tested before the study of clinical samples. To show
that vitreous or aqueous humor was not inhibitory to DNA extraction and PCR,
50-l samples of normal (not infected) vitreous and normal aqueous humor were

VOL. 39, 2001

OCULAR FUNGAL PATHOGEN IDENTIFICATION BY ITS2 TYPING

spiked with 1 l of C. albicans culture (10 cells) as an internal positive control.


DNA was extracted as described above, and PCR was carried out.
(v) DNA sequencing of PCR products. Amplified DNA from PCR was purified
using the GeneClean II kit (Bio 101, Inc., Carlsbad, Calif.) as specified by the
manufacturer and directly cycle sequenced in both directions using the BigDye
terminators Ready Reaction Kit (PE Applied Biosystems, Foster City, Calif.) on
an ABI Prism automated DNA sequencer (model 377, version 2.1.1; Applied
Biosystems Warrington, United Kingdom). The primers used were ITS4 and ITS86.
(vi) Data analysis. The PCGENE program was used to ascertain the specificity
of the method, including a large number of fungi. Ocular pathogenic fungi for
which the rDNA sequence is available in GenBank were assayed for selected
primers hybridization using this program. PCGENE facilitates the positive or
negative theoretical union of primers to the sequence target. After clustal alignment of the selected sequences, fragment sizes were manually calculated. ITS2/
5.8S rDNA sequences were analyzed by using the BLAST alignment program of
the GenBank database (National Institutes of Health). The computer alignment
provides a list of matching organisms, ranked in order of similarity between the
unknown sequence and the sequence of the corresponding organism from the
database.

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-

FIG. 2. (A) Sensitivity of the first PCR (ITS1-ITS4 primer pair)


with C. albicans genomic DNA. M, ladder marker GeneRuler 100bp
DNA Ladder Plus (500 bp, black triangle); C, negative control
(ddH2O). (B) Sensitivity of the seminested PCR (ITS4-ITS86 primer
pair) performed with 1 l of the first-round product of C. albicans
PCR. M, ladder marker pBR322 DNA/BsuRI (434 bp, white triangle;
267 bp, black triangle); C, negative control (ddH2O). (C) Sensitivity
of the first PCR (ITS1-ITS4 primer pair) with A. fumigatus genomic
DNA. M, ladder marker GeneRuler 100bp DNA Ladder Plus (500 bp,
black triangle); C, negative control (ddH2O). (D) Sensitivity of the
seminested PCR (ITS4-ITS86 primer pair) performed with 1 l of the
first-round product of A. fumigatus PCR. M, ladder marker pBR322
DNA/BsuRI (434 bp, white triangle; 267 bp, black triangle); C,
negative control (ddH2O).

ITS4 and ITS86-ITS4 primer pairs with genomic DNA isolated


from human leukocytes or from any of the following bacteria:
S. epidermidis, E. coli, S. aureus, P. aeruginosa, and S. pneumoniae (data not shown).
Other fungi reported in the reference list as ocular pathogens were tested with the PCGENE program to ascertain the
specificity of this method (Table 1). The sizes of the fragments
obtained were in agreement with those obtained by PCR.
(ii) PCR sensitivity. The sensitivity was estimated using two
kinds of samples: DNA dilutions (DNA was extracted from a
culture and subsequently diluted) and culture dilutions (serial
dilutions of cells were prepared and DNA extracted from each
culture).
For C. albicans DNA dilutions, the sensitivity of the first
PCR was found routinely (more than three times) to be 1 to 10
fg (Fig. 2A). The seminested PCR, performed with 1 l from
the first PCR, was positive in all the DNA dilutions (Fig. 2B).
The sensitivity for the mold A. fumigatus was found to be 10 to
100 fg (Fig. 2C). The second round of PCR markedly improved
this sensitivity to 1 fg, similar to the results obtained with C.
albicans (Fig. 2D).
Using cell dilutions, the sensitivity of the PCR was routinely
found to be 1 to 10 organisms (Fig. 3A and B). However, for A.
fumigatus the sensitivity was lower; the first PCR was positive
only when carried out with samples containing 10 to 102 organisms, but with semnested PCR the sensitivity improved to
less than 10 organisms (Fig. 3C and D).

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FIG. 1. (A) Specificity of the first PCR (ITS1-ITS4 primer pair)


with genomic DNA. M, ladder marker (GeneRuler 100bp DNA Ladder Plus) (800 bp; white triangle; 500 bp; black triangle); C: negative
control (double-distilled H2O [dd H2O]). (B) Seminested PCR product (ITS86-ITS4 primer pair). M, ladder marker pBR322 DNA/BsuRI
(234 bp, white triangle; 213 bp; black triangle). The lanes are the same
as in panel A except as indicated. C (1st PCR); negative-control
sample from the first-round amplification; C (2nd PCR), negative
control (ddH2O).

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(iii) Detection of PCR inhibitors in ocular samples. The


expected amplification products were obtained indicating that
no PCR inhibitors were present in the clinical samples (aqueous humor and vitreous) after DNA extraction.
Ocular samples. Six cases of endophthalmitis and three
cases of keratitis were analyzed by molecular and culture methods. In the six cases of endophthalmitis, six aqueous samples
and two vitreous samples were taken. Table 2 shows the results
of Grams stain, culture and PCR of all ocular samples. Samples from patients 2, 5, and 8 were PCR negative with fungal
primers and positive with bacterial primers. The sample from
patient 2 was culture positive for coagulase-negative staphylococci; the patient was successfully treated and responded well
to antibiotic therapy. The sample from patient 5 was PCR
positive with bacterial and P. acnes primers and culture positive for P. acnes. The patient underwent anterior vitrectomy
with intravitreal injection of antibiotics. Clinical and visual
improvement was rapid. Patient 8 is still under antibiotic treatment. The sample from patient 4 was negative for both PCR
and culture analysis. The patient is under clinical observation,
and the case is being reviewed every 3 months. The samples
from patients 3 and 7 were bacterial PCR negative and fungal
PCR positive (Fig. 4). The sequence analysis and the culture
showed a C. parapsilopsis infection. Patient 3 successfully finished the antifungal treatment (fluconazole), and patient 7 is
still under fluconazole treatment. Corneal samples from patients 1, 6, and 9 were positive with fungal primers (Fig. 4). The
sample from one of these patients (patient 1) was also positive
by culture, and fungi were detected in Grams stain; the sample
from patient 9 was positive by culture and not detectable by

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.

Grams stain; the sample from patient 6 was negative by both


techniques (culture and Gram stain visualization) and a nested
PCR was necessary to detect the fungal DNA (Fig. 4). Patients
1 and 6 responded well to treatment with antifungal agents,
and patient 9 is still under antifungal treatment with fluconazole and amphotericin B. Patient 10 had keratitis and was
being treated at Mo
stoles Hospital (Madrid). Microscopic visualization and conventional culture were positive for fungi,
and Scedosporium apiospermum was identified (E. Amor, personal communication). The DNA was extracted from culture,
and its ITS/5.8S rDNA sequence confirmed the identification
as S. apiospermum. Although DNA extraction from corneal
samples was not done, this case was also considered interesting
for the assessment of the technique.
Microscopic fungal visualization (Gram stains) was negative
for three of the six ocular samples (Table 2). Only the corneal
sample from patient 6 and the aqueous sample from patient 8
were negative by culture and positive by PCR. The other samples showed the same results by culture and by PCR. However,
cultures needed an average of 6 to 7 days to grow, while the
PCR results were obtained in 6 to 8 h.

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FIG. 3. (A) Sensitivity of the first PCR (ITS1-ITS4 primer pair)


with C. albicans cells. M, ladder marker GeneRuler 100bp DNA Ladder Plus (500 bp, black triangle); C, negative control (ddH2O). (B)
Sensitivity of the seminested PCR (ITS4-ITS86 primer pair) performed with 1 l of the first-round product of C. albicans PCR. M,
ladder marker pBR322 DNA/BsuR1 (434 bp, white triangle; 267 bp,
black triangle); C, negative control (ddH2O). (C) Sensitivity of the
first PCR (ITS1-ITS4 primer pair) with A. fumigatus cells. M, ladder
marker GeneRuler 100bp DNA Ladder Plus (500 bp, black triangle);
C, negative control (ddH2O). (D) Sensitivity of the seminested PCR
(ITS4-ITS86 primer pair) performed with 1 l of the first-round product of A. fumigatus PCR. M, ladder marker pBR322 DNA/BsuRI (434
bp, white triangle; 267 bp, black triangle); C, negative control
(ddH2O).

VOL. 39, 2001

OCULAR FUNGAL PATHOGEN IDENTIFICATION BY ITS2 TYPING

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TABLE 2. Gram stain, culture, and PCR results with samples from 10 patients with a clinical diagnosis of endophthalmitis or keratitisa
Patient

Sample

Gram stain resultb

Culture result

Bacterial PCR/P. acnes PCR resultc

Fungal PCR result/homology sequence

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

ND, not done.


, insufficient sample.
Where just one result is shown, it is the result of the bacterial PCR test (the second test was unnecessary).

DNA sequencing. DNA database comparison of the DNA


sequences obtained with the full-sequence ITS2 and partialsequence 5.8S rDNA from the ocular samples demonstrated
that they were derived from the fungal ITS regions. Two of
them were identical to the C. parapsilosis ITS2/5.8S rDNA
region, and one each were identical to the A. niger, A. fumigatus, Alternaria alternata, and Scedosporium apiospermum ITS2/
5.8S rDNA region (Table 2).
DISCUSSION
In this report, an optimized rapid technique for the detection and identification of fungi in ocular samples is presented.
It consists of an inexpensive and rapid method of DNA extraction and a sensitive and precise method of identification of
fungal pathogens based on amplification of ITS2/5.8S rDNA
and molecular typing.
A good DNA extraction method is critical for PCR detection
to avoid the possibilities of false-negative results. We tested
some commercial kits (Instagene; Bio-Rad Laboratories, Hercules, Calif., and Mo-Bio Laboratories, Inc., Solana Beach,
Calif.) for rapid extraction of DNA, but the result was not
satisfactory for all the tested fungal strains (A. niger or C.
neoformans). Similar results with the use of other commercial
kits, except for a QIAmp Tissue kit, for fungal DNA extraction
have been previously described (25). This protocol applied to
vitreous ocular samples provides high-quality DNA that is devoid of PCR-inhibiting substances. The DNA loss is minimal
because we have been able to amplify DNA from 1 to 10
microorganisms (C. albicans) and from 10 to 100 microorganisms (A. fumigatus). Additionally, this procedure is rapid, technically simple, broadly applicable, and inexpensive.
rRNA genes are highly conserved in all fungal species tested
to date. The use of rRNA genes for identification of fungal
species is based on the detection of conserved sequences in the
rDNA genes. Our results showed high fungal specificity with
the selected primers. All fungal strains were PCR positive, and
all negative controls (human and bacterial DNA) were PCR
negative. This same protocol of extraction and amplification of
DNA from ocular samples was performed in an animal fungal
infection model (11). In this work, the fungal infection was
induced in one eye of each of five rabbits (New Zealand)
with C. albicans, C. parapsilopsis, A. niger, A. fumigatus, and

F. oxyosporum, The DNA sequence target was detected in all


infection samples studied.
The primers used for PCR amplification were checked and
found to be specific for fungal rDNA; they did not target
prokaryotic rDNA sequences. Moreover, they targeted all the
rDNA sequences from ocular pathogenic fungi available in
database.
As discussed above, the sensitivity for the first amplification
of the ITS/5.8S rDNA region was 1 fg for C. albicans and 10 fg
for A. fumigatus in water dilutions of DNA. Assuming a total
DNA content of 37 fg per organism (38), this amount is equivalent to less than one C. albicans cell. This is in agreement with
the fact that rRNA genes are multiple-copy genes, with 100 or
more copies within the fungal genome (32), making them ideal
targets for PCR amplification and permitting the amplification
from a very small number of microorganisms.
When culture dilutions were used as targets for amplification, 1 to 10 organisms of C. albicans and fewer than 100
organisms of A. fumigatus could be detected. As shown in Fig.
2 and 3, the intensity of the band corresponding to the PCR
carried out with 10 to 100 fg of genomic C. albicans DNA is
similar to the intensity of the band corresponding to the PCR
with 1 to 10 microorganisms (the total DNA content is of 37 fg
per organism) (38). For A. fumigatus, the intensity of the band
corresponding to the PCR performed with 100 fg of DNA was
similar to that obtained with 1 to 10 microorganisms (total
DNA of this mold could be estimated at approximately 35 Mb
[100 fg]) (24). This means that there was no significant DNA
loss during the DNA extraction. Nevertheless, to make sure, a
large number of experiments would be necessary because the
range given for the DNA amount and the range given for cell
numbers in these preparations were not exactly the same.
Infectious agents were detected by PCR in eight of nine clinical
ocular infections; five of them were positive by amplification with
fungal primers, and three were positive by amplification with
bacterial primers. In seven of the cases, the pathogen could also
be retrieved by cultivation (two bacteria and five fungi). However,
while the PCR result was obtained in a few hours, cultures
needed 5 to 6 days to grow and 2 to 3 days for identification.
When PCR was followed by sequencing of the PCR product,
the total identification time was 24 h, still significantly shorter
than that needed for cultured-based identification.

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2878

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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The advantage of rapidly ascertaining the fungal or bacterial


origin of the infection is complemented by the rapid identification of the fungus itself. For example, patient 1 had received
intravitreal amphotericin B as the first treatment. The identification of C. parapsilosis as responsible for the infection permitted the treatment to be changed from amphotericin B to
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studies with a larger number of clinical samples are necessary
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OCULAR FUNGAL PATHOGEN IDENTIFICATION BY ITS2 TYPING