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Previous Article | Next Article 
Journal of Clinical Microbiology, August 2001, p. 2873-2879, Vol. 39, No. 8
Departamento de Biología Molecular,
Instituto Oftalmológico de Alicante, 03015 Alicante,1 and Div.
Microbiología,2 and
Patología y Cirugía-Div.
Oftalmología,3 Universidad Miguel
Hernández, 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
(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.
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.
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% [wt/vol] glucose, 1%
[wt/vol] peptone) supplemented with chloramphenicol (1 mg
liter
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.8.2873-2879.2001
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
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
1), subcultured onto Sabouraud dextrose agar slants,
and kept at 4°C.
TABLE 1.
Strains and source of ocular isolates analyzed by PCR
amplification of rDNA
(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 30°C. 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
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-mercaptoethanol). The lysate was extracted with
phenol-chloroform-isoamyl alcohol (25:24:1, vol/vol/vol). Then, 65 µl
of 3 M sodium acetate and 75 µl of 1 M NaCl were added to 350 µl 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 from C. albicans
and A. fumigatus were prepared to different concentrations
(10 ng to 1 fg per 10 µl) 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, 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 (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, Barcelona, Spain). The 50-µl PCR mixture contained 10 µl of DNA template, 6 µl of 25 mM MgCl2, 5 µl of PCR buffer without MgCl2; 200 µM 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 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 1 µ1 of first-round product in 50 µl of PCR reaction mixture (6 µl of 25 mM MgCl2, 5 µl of PCR buffer without MgCl2, 200 µM 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 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 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 µl 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 µl) 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 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 Oftalmológico 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 µl of aqueous fluid was withdrawn using a 30-gauge needle with a
limbal paracentesis. Vitreous samples (200 µl) 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.
(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
Löwenstein-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 µl of
sterile water in a 1.5-ml sterile Eppendorf tube. Two aliquots of 50 µl were taken from each sample and stored at 20°C.
(iii) Fungal DNA extraction.
A 50-µl 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 10 µl 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-µl samples of normal (not infected) vitreous and normal aqueous humor were spiked with 1 µl 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.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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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).
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(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 µl 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).
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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
Gram's 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 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 fluconazole and amphotericin B. Patient 10 had keratitis and was being treated at Móstoles 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.
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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. fumigatus, Alternaria alternata, and Scedosporium apiospermum ITS2/5.8S rDNA region (Table 2).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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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.
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 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 identification. 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 significantly less information is obtained. Other molecular techniques 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. Therefore 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 possible involvement of these genera as pathogens. In contrast, the amplification and typing of the ITS region eliminates this requirement. 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 standard 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.
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ACKNOWLEDGMENTS |
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This work was supported by grant IMTEIA/1998/210 from the IMPIVA (Generalitat Valenciana, Spain) and a grant from Instituto Oftalmológico 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 Hernández for her English language corrections; and Josefa Antón for scientific suggestions. We also thank Elisa Amor from Móstoles 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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FOOTNOTES |
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* Corresponding author. Mailing address: Dpto. Biología Molecular, Instituto Oftalmológico de Alicante, Avenida de Denia no. 111, 03015 Alicante, Spain. Phone: 34 965 154062. Fax: 34 965 160468. E-mail: cferre{at}umh.es.
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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