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Detection and Identification of Fungal Pathogens by PCR and by ITS2 and 5 8S Ribosomal DNA Typing in Ocular Infections

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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,

2

SUSANA FRASE´S,

2

EMILIA MULET,

3

JOSE´ L. ABAD,

1AND

JORGE L. ALIO

´

1,3

Departamento de Biologı´a Molecular, Instituto Oftalmolo´gico de Alicante, 03015 Alicante,

1

and Div. Microbiologı´a,

2

and Patologı´a y Cirugı´a-Div. Oftalmologı´a,

3

Universidad 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 5106cells/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, 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 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 10␮l) 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 10␮l of DNA template, 6␮l of

25 mM MgCl2, 5␮l of PCR buffer without MgCl2; 200␮M 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 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 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, 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⫻

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 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 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, 50␮l of aqueous humor or 50␮l 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 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⫺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 10␮l 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 1␮l 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

2

or-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

2

O [dd H

2

O]). (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

st

PCR); negative-control

sample from the first-round amplification; C

(2

nd

PCR), negative

control (ddH

2

O).

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

2

O). (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

2

O). (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

2

O). (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

2

O).

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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

(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

2

O). (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

2

O). (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

2

O). (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

2

O).

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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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

a

Patient 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.

b, 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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Figure

TABLE 1. Strains and source of ocular isolates analyzed by PCRamplification of rDNA
FIG. 1. (A) Specificity of the first PCR (ITS1-ITS4 primer pair)with genomic DNA. M, ladder marker (GeneRuler 100bp DNA Lad-
FIG. 4. (A) First PCR (ITS1-ITS4 primer pair) from different oc-ular samples. M, ladder marker GeneRuler 100bp DNA Ladder Plus
TABLE 2. Gram stain, culture, and PCR results with samples from 10 patients with a clinical diagnosis of endophthalmitis or keratitisa

References

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