Copyright © 2001, American Society for Microbiology. All Rights Reserved.
Detection and Identification of Fungal Pathogens by PCR and by ITS2
and 5.8S Ribosomal DNA Typing in Ocular Infections
CONSUELO FERRER,
1* FRANCISCA COLOM,
2SUSANA FRASE´S,
2EMILIA MULET,
3JOSE´ L. ABAD,
1ANDJORGE L. ALIO
´
1,3Departamento de Biologı´a Molecular, Instituto Oftalmolo´gico de Alicante, 03015 Alicante,
1and Div. Microbiologı´a,
2and Patologı´a y Cirugı´a-Div. Oftalmologı´a,
3Universidad Miguel Herna´ndez, 03550 Alicante, Spain
Received 14 March 2001/Returned for modification 4 April 2001/Accepted 3 June 2001
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
(endoph-thalmitis 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.
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
con-firmed by obtaining intraocular (aqueous or vitreous)
speci-mens 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
crit-ical element for an effective treatment of ocular infections.
This has led to the development of culture-independent
diag-nostic 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
uni-versal fungal PCR primers have been developed for
broaden-ing 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
multi-copy 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
tran-scriptional 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
tran-scribed 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
tech-niques (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
pa-tients 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.
MATERIALS AND METHODS
Standard fungal isolates. (i) Strains.Clinical and standard isolates of
Aspergil-lus, Candida, Fusarium, Scedosporium, Alternaria, andCryptococcuswere used in this study (Table 1). Strains were cultured on Sabouraud dextrose broth (2%
* Corresponding author. Mailing address: Dpto. Biologı´a
Molecu-lar, 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.
2873
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[wt/vol] glucose, 1% [wt/vol] peptone) supplemented with chloramphenicol (1
mg liter⫺1), subcultured onto Sabouraud dextrose agar slants, and kept at 4°C.
(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 con-taining 0.5 ml of Sabouraud dextrose broth supplemented with chloramphenicol and incubated overnight in an orbital shaker at 150 rpm and 30°C. Thereafter, fungal cultures were adjusted photometrically (absorbance at 530 nm;
McFar-land 0.5 standard) to a concentration of 1⫻106to 5⫻106cells/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 ofCandida
albicansandAspergillus fumigatus(106to 100cells) were prepared to test the
sensitivity and specificity of the assay. The fungal suspensions with
predeter-mined concentrations were centrifuged at 5,000⫻g, and then the pellet was
frozen at⫺20°C for 1 h and incubated at 65°C for 1 h in 0.5 ml of extraction
buffer (50 mM Tris-HCl, 50 mM EDTA, 3% sodium dodecyl sulfate, 1% 2-mer-captoethanol). The lysate was extracted with phenol-chloroform-isoamyl alcohol
(25:24:1, vol/vol/vol). Then, 65l of 3 M sodium acetate and 75l of 1 M NaCl
were added to 350l of the supernatant and the resulting volume was incubated
at 4°C 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 fromC. albicansandA. fumigatuswere
pre-pared to different concentrations (10 ng to 1 fg per 10l) and stored at⫺20°C.
(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,
andStreptococcus pneumoniaefrom the Spanish Type Culture Collection. Bac-terial DNA was isolated by using the InstaGene Matrix.
(iv) PCR assay.Extracted DNA was amplified using a RoboCycler 96
tem-perature 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
amplifi-cation were ITS1 (5⬘TCC GTA GGT GAA CCT GCG G 3⬘), which hybridizes
at the end of 18S rDNA, and ITS4 (5⬘TCC TCC GCT TAT TGA TAT GC 3),
which hybridizes at the beginning of 28S rDNA (44) (Life Technologies,
Barce-lona, Spain). The 50-l PCR mixture contained 10l of DNA template, 6l of
25 mM MgCl2, 5l of PCR buffer without MgCl2; 200M each deoxynucleoside
triphosphate, 25 pmol of each primer, and 1 U ofTaqDNA polymerase (Biotools
B&M Labs, S.A., Madrid, Spain). Reactions involved 1 cycle at 95°C for 5 min, followed by 35 cycles with a denaturation step at 95°C for 30 s, an annealing step at 55°C for 1 min, and an extension step at 72°C for 1 min, followed by 1 cycle at 72°C for 6 mins.
(b) Seminested amplification.For the second amplification, the primers used
were ITS86 (5⬘GTG 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 11 of first-round product in
50l of PCR reaction mixture (6l of 25 mM MgCl2, 5l of PCR buffer without
MgCl2, 200M each deoxynucleoside triphosphate, 50 pmol of primer ITS4, and
100 pmol of primer ITS86, and 1 U ofTaqDNA polymerase (Biotools B&M
Labs). Reactions involved 1 cycle at 95°C for 5 min, followed by 30 cycles with a denaturation step at 95°C for 30 s, an annealing step at 55°C for 30 s, and an extension step at 72°C for 30 s, followed by 1 cycle at 72°C for 6 min.
(c) Negative controls.Two negative controls were included in the first
ampli-fication: 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, 1l 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 (10l) of each amplified
product were electrophoretically separated in a 2% agarose gel in 1⫻
Tris-borate-EDTA buffer and visualized using ethidium bromide under UV illumi-nation. 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
scrap-ings 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 scrap-ings was approved by the Institutional Review Board at the Instituto Oftalmo-lo´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 200l of aqueous fluid was withdrawn
using a 30-gauge needle with a limbal paracentesis. Vitreous samples (200l)
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 4°C. One portion was immediately examined by conventional microbiological diagnostic tests, and the other was frozen at
⫺20°C until processed by PCR.
For the microbiological diagnostic test, 50l of aqueous humor or 50l of
vitreous was cultured at 30°C in Sabouraud’s dextrose agar or at 37°C in thio-glycolate broth, blood agar, chocolate agar, CLED agar, or MacConkey agar. Bacteria were identified by the API Staph and API 20A systems (Biomeriux, bioMe´rieux Sa, Marcy L Etoile, France). Yeasts were identified by the Auxacolor system (Sanofi Diagnostics Pasteur, Inc, Marnes-la-Coquette, France), and fila-mentous fungi were differentiated by isolation in Sabouraud dextrose agar plus chloramphenicol and morphological examination of a macroscopic and micro-scopic 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 100l of sterile water in a
1.5-ml sterile Eppendorf tube. Two aliquots of 50 l were taken from each
sample and stored at⫺20°C.
(iii) Fungal DNA extraction.A 50-l volume of each ocular sample was frozen
for at least 1 h at⫺20°C. The DNA extraction was performed as described for
the standard fungi isolates. DNA was diluted in 10l of sterile water.
(iv) PCR assay.The PCR for fungal DNA detection was performed as
de-scribed for the standard fungal isolates. The bacterial PCR and specific
Propi-onibacterium acnesPCR 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,
[image:2.612.52.293.91.296.2]50-l samples of normal (not infected) vitreous and normal aqueous humor were
TABLE 1. Strains and source of ocular isolates analyzed by PCR
amplification of rDNA
Straina
Predicted size (bp) of PCR product
with primers:
ITS1-ITS4 ITS86-ITS4
Alternaria alternata
IOA-K3
570
292
Aspergillus flavus
CECT 2684
595
300
Aspergillus fumigatus
CECT 2071/IOA-K1
596
299
Aspergillus niger
CECT 2574/IOA-K2
599
300
Aspergillus terreus
CECT 2748
608
308
Candida albicans
CECT 1472
536
282
Candida parapsilosis
ATCC 22019/IOA-E1, IOA-E2 520
255
Candida tropicalis
CECT 1005
524
270
Candida glabrata
CECT 1448
820
360
Candida kruseii
ATCC 6258
510
294
Fusarium oxysporum
CECT 2154
544
283
Fusarium solani
CECT 2199
569
286
Cryptococcus neoformans
var.
neoformans
SCNC SP1
556
320
var.
gatti
SCNC C48
556
320
Scedosporium apiospermum
HMM-K1
611
329
aIOA; ocular isolates obtained from the Instituto Oftalmolo´gico de Alicante,
Alicante, Spain; HMM, Ocular isolates obtained from Mo´stoles Hospital,
Ma-drid, Spain; SCNC, Clinical isolates obtained from SpanishCryptococcus
neofor-mansCollection, Mycology Laboratory, Universidad Miguel Herna´ndez,
Ali-cante, Spain; CECT, Spanish Type Culture Collection, Valencia, Spain; ATCC, Americans Type Culture Collection. Rockville, Md.
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spiked with 1l ofC. albicansculture (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 align-ment of the selected sequences, fragalign-ment 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
amplifi-cation 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
approxi-mately 550 bp was obtained. After the second round of
ampli-fication, the fragment obtained was about 280 bp (Fig. 1).
No amplification products were detected by using the
ITS1-ITS4 and ITS86-ITS1-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.
pneu-moniae
(data not shown).
Other fungi reported in the reference list as ocular
patho-gens 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 10
2or-ganisms, but with semnested PCR the sensitivity improved to
less than 10 organisms (Fig. 3C and D).
FIG. 1. (A) Specificity of the first PCR (ITS1-ITS4 primer pair)
with genomic DNA. M, ladder marker (GeneRuler 100bp DNA
Lad-der Plus) (800 bp; white triangle; 500 bp; black triangle); C
⫺
: negative
control (double-distilled H
2O [dd H
2O]). (B) Seminested PCR
prod-uct (ITS86-ITS4 primer pair). M, ladder marker pBR322 DNA/
Bsu
RI
(234 bp, white triangle; 213 bp; black triangle). The lanes are the same
as in panel A except as indicated. C
⫺
(1
stPCR); negative-control
sample from the first-round amplification; C
⫺
(2
ndPCR), negative
control (ddH
2O).
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
(ddH
2O). (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/
Bsu
RI (434 bp, white triangle;
267 bp, black triangle); C
⫺
, negative control (ddH
2O). (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 (ddH
2O). (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/
Bsu
RI (434 bp, white triangle; 267 bp, black triangle); C
⫺
,
negative control (ddH
2O).
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[image:3.612.57.295.73.336.2](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
(aque-ous humor and vitre(aque-ous) after DNA extraction.
Ocular samples.
Six cases of endophthalmitis and three
cases of keratitis were analyzed by molecular and culture
meth-ods. In the six cases of endophthalmitis, six aqueous samples
and two vitreous samples were taken. Table 2 shows the results
of Gram’s stain, culture and PCR of all ocular samples.
Sam-ples 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
staphylo-cocci; 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
posi-tive 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
treat-ment. 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
fin-ished the antifungal treatment (fluconazole), and patient 7 is
still under fluconazole treatment. Corneal samples from
pa-tients 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 Gram’s stain; the sample
from patient 9 was positive by culture and not detectable by
Gram’s 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
flucon-azole and amphotericin B. Patient 10 had keratitis and was
being treated at Mo´stoles Hospital (Madrid). Microscopic
vi-sualization and conventional culture were positive for fungi,
and
Scedosporium apiospermum
was identified (E. Amor,
per-sonal 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
sam-ples 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.
FIG. 3. (A) Sensitivity of the first PCR (ITS1-ITS4 primer pair)
with
C. albicans
cells. M, ladder marker GeneRuler 100bp DNA
Lad-der Plus (500 bp, black triangle); C
⫺
, negative control (ddH
2O). (B)
Sensitivity of the seminested PCR (ITS4-ITS86 primer pair)
per-formed with 1
l of the first-round product of
C. albicans
PCR. M,
ladder marker pBR322 DNA/
Bsu
R1 (434 bp, white triangle; 267 bp,
black triangle); C
⫺
, negative control (ddH
2O). (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 (ddH
2O). (D) Sensitivity of the seminested PCR
(ITS4-ITS86 primer pair) performed with 1
l of the first-round
prod-uct of
A. fumigatus
PCR. M, ladder marker pBR322 DNA/
Bsu
RI (434
bp, white triangle; 267 bp, black triangle); C
⫺
, negative control
(ddH
2O).
FIG. 4. (A) First PCR (ITS1-ITS4 primer pair) from different
oc-ular 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/
Bsu
RI (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.
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[image:4.612.314.550.83.401.2]DNA sequencing.
DNA database comparison of the DNA
sequences obtained with the full-sequence ITS2 and
partial-sequence 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.
fumiga-tus,
Alternaria alternata
, and
Scedosporium apiospermum
ITS2/
5.8S rDNA region (Table 2).
DISCUSSION
In this report, an optimized rapid technique for the
detec-tion and identificadetec-tion of fungi in ocular samples is presented.
It consists of an inexpensive and rapid method of DNA
extrac-tion and a sensitive and precise method of identificaextrac-tion 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,
Her-cules, 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
de-void 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
microorgan-isms (
A. fumigatus
). Additionally, this procedure is rapid,
tech-nically 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
equiv-alent 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
amplifica-tion, 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.
[image:5.612.56.551.82.213.2]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.
TABLE 2. Gram stain, culture, and PCR results with samples from 10 patients with a clinical diagnosis of endophthalmitis or keratitis
aPatient Sample Gram stain resultb Culture result Bacterial PCR/P. acnesPCR resultc Fungal PCR result/homology sequence
1
Corneal Scrape
Positive
Positive (2 days)
ND
Positive/
A. fumigatus
2
Aqueous
ND*
Positive (3 days)
Positive/negative
Negative
3
Aqueous
ND*
Positive (6 days)
Negative
Positive/
C. parapsilosis
4
Aqueous
ND*
Negative
Negative
Negative
5
Aqueous
ND*
Positive (13 days)
Positive/positive
Negative
5
Vitreous
Positive
Positive (7 days)
Positive/positive
Negative
6
Corneal scrape
Negative
Negative
ND
Positive/
A. niger
7
Aqueous
ND*
Negative
Negative
Negative
7
Vitreous
Negative
Positive (7 days)
Negative
Positive/
C. parapsilosis
8
Aqueous
ND*
Negative
Positive/negative
Negative
9
Corneal scrape
Negative
Positive (7 days)
ND
Positive/
Alternaria alternata
10
Culture of corneal scrape
Positive
Positive
ND
Positive/
S. apiospermum
aND, not done.
bⴱ, insufficient sample.
cWhere just one result is shown, it is the result of the bacterial PCR test (the second test was unnecessary).
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The advantage of rapidly ascertaining the fungal or bacterial
origin of the infection is complemented by the rapid
identifi-cation of the fungus itself. For example, patient 1 had received
intravitreal amphotericin B as the first treatment. The
identi-fication of
C. parapsilosis
as responsible for the infection
per-mitted the treatment to be changed from amphotericin B to
fluconazole (4, 19).
Analysis of sequences (5.8S/ITS region) from the database
confirmed that this method can be used to differentiate fungi at
the species level. Some studies show that fungal strains can be
distinguished on the basis of the size of the ITS/5.8S fragment
(6, 41) and primary structural differences in the rDNA spacer
regions (10, 46). However, although yeast demonstrated a
higher level of interspecies variability compared to other fungi,
size determination based on agarose gel electrophoresis is not
precise enough to unmistakeably confirm the species
identifi-cation. Table 1 shows that fragment sizes are very similar and
therefore very difficult to differentiate in an agarose gel. If the
size is determined by capillary electrophoresis (41), the time
required is similar to that needed in sequencing and
signifi-cantly less information is obtained. Other molecular
tech-niques proposed for fungal identification, such as the use of
restriction fragment length polymorphism analysis of ITS/5.8S
fragments, hybridization with a specific probe, and the specific
nested PCR (10, 35, 46), could be useful to confirm a specific
fungal infection, for example in endophthalmitis (frequently
produced by
Candida
,
Aspergillus
and
Fusarium
). However, the
range of fungi capable of causing keratitis is significantly wider
than that of fungi capable of causing endophthalmitis.
There-fore a large number of species causing infection could remain
unidentified by these molecular methods. Specifically, in the
course of this study, the identification for patients 9 and 10
(
Alternaria
and
Scedosporium
) would be rather complicated
and time-consuming because of the need to consider the
pos-sible involvement of these genera as pathogens. In contrast, the
amplification and typing of the ITS region eliminates this
re-quirement. In addition, the small size of the fragment permits
its sequencing in both directions at once, and the obtained
sequence gives enough information to identify the fungal species.
This method proved to be reproducible and very useful for
easy and rapid identification and classification of all the species
included in the present work. This is the first time that these
rDNA-specific primers have been successfully used for fungal
detection and identification in ocular samples. This PCR-based
method promises to be very effective for the diagnosis of fungal
ocular infections in the clinical setting. Compared with
stan-dard laboratory techniques, it offers a significant reduction of
the time required to establish the diagnosis. However, further
studies with a larger number of clinical samples are necessary
to assess the efficacy of the method.
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 Herna´ndez for her English
lan-guage corrections; and Josefa Anto´n for scientific suggestions. We also
thank Elisa Amor from Mo´stoles Hospital (Madrid, Spain) for her
col-laboration with the study of the
Scedosporium apiospermum
strain and
for providing information on the keratitis case associated with this strain.
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