JOURNAL OF CLINICAL MICROBIOLOGY,0095-1137/01/$04.0010 DOI: 10.1128/JCM.39.8.2873–2879.2001

Aug. 2001, p. 2873–2879 Vol. 39, No. 8

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Detection and Identification of Fungal Pathogens by PCR and by ITS2and 5.8S Ribosomal DNA Typing in Ocular InfectionsCONSUELO FERRER,1* FRANCISCA COLOM,2 SUSANA FRASES,2 EMILIA MULET,3

JOSE L. ABAD,1 AND JORGE L. ALIO1,3

Departamento de Biologıa Molecular, Instituto Oftalmologico de Alicante, 03015 Alicante,1 and Div. Microbiologıa,2

and Patologıa y Cirugıa-Div. Oftalmologıa,3 Universidad Miguel Hernandez, 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/5.8Sribosomal 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 PCRand seminested PCR to detect fungal DNA. Fifty strains of 12 fungal species (yeasts and molds) were used totest the selected primers and conditions of the PCR. PCR and seminested PCR of this region were carried outto evaluate the sensitivity and specificity of the method. It proved possible to amplify the ITS2/5.8S region ofall the fungal strains by this PCR method. All negative controls (human and bacterial DNA) were PCRnegative. The sensitivity of the seminested PCR amplification reaction by DNA dilutions was 1 organism perPCR, and the sensitivity by cell dilutions was fewer than 10 organisms per PCR. Intraocular sampling orcorneal scraping was undertaken for all patients with suspected infectious endophthalmitis or keratitis(nonherpetic), respectively, between November 1999 and February 2001. PCRs were subsequently performedwith 11 ocular samples. The amplified DNA was sequenced, and aligned against sequences in GenBank at theNational Institutes of Health. The results were PCR positive for fungal primers for three corneal scrapings, oneaqueous sample, and one vitreous sample; one of them was negative by culture. Molecular fungal identificationwas successful in all cases. Bacterial detection by PCR was positive for three aqueous samples and one vitreoussample; one of these was negative by culture. Amplification of ITS2/5.8S rDNA and molecular typing showspotential as a rapid technique for identifying fungi in ocular samples.

The microbiological spectrum of infectious endophthalmitisshows that the percentage of isolates that are fungi is 8 to18.5% (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 microbiologicaltests are positive in only 54 to 69% of endophthalmitis cases(13, 22, 23) (by culture) and 80% (8) of keratitis cases (byGram and Giemsa stains and culture). In fungal infections,even when positive, results usually take longer than a weekbecause these organisms are difficult to identify and/or areslow-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 withuniversal primers for bacterial DNA in ocular samples (5, 16,20, 21, 26, 27, 34, 36, 40) have recently been developed. Fordetection of fungal pathogens, multicopy gene targets havebeen 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 onfungal DNA detection in ocular samples have been performed(3, 15, 17, 35); the small number of conidia in the samples, thedifficulty of DNA extraction (25, 43) (some filamentous fungihave a sturdy cell wall which is resistant to standard DNA

extraction procedures for yeast and bacteria), and the presenceof PCR inhibitors in human specimens (45) are some of thedifficulties with fungal detection in ocular samples. The idealmarker to detect a fungal infection should be present in allfungal genera (but should contain enough internal variation inits 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 inhigh copy number and the sensitivity of their detection may bedramatically 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 28Sribosomal DNA (rDNA) gene subunits are intergenic tran-scribed spacer regions (ITS1 and ITS2) that are not translatedinto rRNA. Although rRNA genes are highly conserved theITS 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 ofdifferent 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 formycological diagnosis, increase the number of ocular samplesfrom which a confirmed diagnosis is made, and identify thecausative fungal agent.

MATERIALS AND METHODS

Standard fungal isolates. (i) Strains. Clinical and standard isolates of Aspergil-lus, Candida, Fusarium, Scedosporium, Alternaria, and Cryptococcus were used inthis study (Table 1). Strains were cultured on Sabouraud dextrose broth (2%

* Corresponding author. Mailing address: Dpto. Biologıa Molecu-lar, Instituto Oftalmologico de Alicante, Avenida de Denia no. 111,03015 Alicante, Spain. Phone: 34 965 154062. Fax: 34 965 160468.E-mail: cferre@umh.es.

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[wt/vol] glucose, 1% [wt/vol] peptone) supplemented with chloramphenicol (1mg liter21), 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 equipmentdesignated 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 chloramphenicoland 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 3 106 to 5 3 106 cells/ml. In the caseof filamentous fungi, conidia were separated from the rest of the mycelium byfiltration through sterile glass wool (28). Tenfold serial dilutions of Candidaalbicans and Aspergillus fumigatus (106 to 100 cells) were prepared to test thesensitivity and specificity of the assay. The fungal suspensions with predeter-mined concentrations were centrifuged at 5,000 3 g, and then the pellet wasfrozen at 220°C for 1 h and incubated at 65°C for 1 h in 0.5 ml of extractionbuffer (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 ml of 3 M sodium acetate and 75 ml of 1 M NaClwere added to 350 ml of the supernatant and the resulting volume was incubatedat 4°C for 30 min. DNA was recovered by isopropanol precipitation and washedwith 70% (vol/vol) ethanol. The concentration was measured by monitoring theUV absorbance at 260 nm (Gene Quant System; Pharmacia, LKB Biochrom).Serial aqueous dilutions of DNA from C. albicans and A. fumigatus were pre-pared to different concentrations (10 ng to 1 fg per 10 ml) and stored at 220°C.

(iii) Negative controls. (a) Extraction of DNA from human leukocytes. HumanDNA from whole blood was isolated by using the InstaGene Matrix (Bio-RadLaboratories, Hercules, Calif.) as specified by the manufacturer.

(b) Extraction from bacteria. A variety of bacterial organisms capable ofproducing ocular infections were used to determine the specificity of the fungalprimers: Staphylococcus epidermidis, Pseudomonas aeruginosa, Escherichia coli,and Streptococcus pneumoniae from 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 conditionsused 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 (59TCC GTA GGT GAA CCT GCG G 39), which hybridizesat the end of 18S rDNA, and ITS4 (59TCC TCC GCT TAT TGA TAT GC 3),which hybridizes at the beginning of 28S rDNA (44) (Life Technologies, Barce-lona, Spain). The 50-ml PCR mixture contained 10 ml of DNA template, 6 ml of

25 mM MgCl2, 5 ml of PCR buffer without MgCl2; 200 mM each deoxynucleosidetriphosphate, 25 pmol of each primer, and 1 U of Taq DNA polymerase (BiotoolsB&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 stepat 55°C for 1 min, and an extension step at 72°C for 1 min, followed by 1 cycleat 72°C for 6 mins.

(b) Seminested amplification. For the second amplification, the primers usedwere ITS86 (59GTG AAT CAT CGA ATC TTT GAA C 3), which hybridizeswith the 5.8S rDNA region (29), and ITS4 (Life Technologies, Barcelona, Spain).Seminested PCR amplification mixtures contained 1 m1 of first-round product in50 ml of PCR reaction mixture (6 ml of 25 mM MgCl2, 5 ml of PCR buffer withoutMgCl2, 200 mM each deoxynucleoside triphosphate, 50 pmol of primer ITS4, and100 pmol of primer ITS86, and 1 U of Taq DNA polymerase (Biotools B&MLabs). Reactions involved 1 cycle at 95°C for 5 min, followed by 30 cycles with adenaturation step at 95°C for 30 s, an annealing step at 55°C for 30 s, and anextension 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. Thesample extraction control consisted of sterile MilliQ water subjected to the sameextraction procedures as the specimens. In the seminested PCR, 1 ml each of thetwo negative control samples from the first-round amplification and a thirdnegative control of sterile water were included.

(v) Detection of the amplified products. Aliquots (10 ml) of each amplifiedproduct were electrophoretically separated in a 2% agarose gel in 13 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 endophthalmitisor keratitis (nonherpetic), respectively, between November 1999 and February2001. Before sampling, informed consent was obtained from all patients. Theprotocol for collection of aqueous samples, vitreous samples, and corneal scrap-ings was approved by the Institutional Review Board at the Instituto Oftalmo-logico de Alicante, Alicante, Spain. This research followed the tenets of theDeclaration of Helsinki at all times.

(ii) Sample collection and culture. (a) Procedure for endophthalmitis cases.The extraocular environment was sterilized with 5% povidone iodine solutionbefore surgery. Approximately 100 to 200 ml of aqueous fluid was withdrawnusing a 30-gauge needle with a limbal paracentesis. Vitreous samples (200 ml)were taken at the time of three-port pars plana vitrectomy. The samples weredivided into two aliquots and transported to the microbiology laboratory and tothe molecular biology laboratory at 4°C. One portion was immediately examinedby conventional microbiological diagnostic tests, and the other was frozen at220°C until processed by PCR.

For the microbiological diagnostic test, 50 ml of aqueous humor or 50 ml ofvitreous 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,bioMerieux Sa, Marcy L Etoile, France). Yeasts were identified by the Auxacolorsystem (Sanofi Diagnostics Pasteur, Inc, Marnes-la-Coquette, France), and fila-mentous fungi were differentiated by isolation in Sabouraud dextrose agar pluschloramphenicol and morphological examination of a macroscopic and micro-scopic characteristics.

(b) Procedure for keratitis cases. Upon completion of the ocular examinationand after instillation of topical anesthetic, a sterile Kimura spatula was used toscrape the area of infection. Scrapings were inoculated into thioglycolate broth,Roiron broth, and Lowenstein-Jensen medium and were placed onto glass slidesfor staining with Gram and Giemsa stains. The PCR sample was obtained byscraping and stirring the spatula for a few seconds in 100 ml of sterile water in a1.5-ml sterile Eppendorf tube. Two aliquots of 50 ml were taken from eachsample and stored at 220°C.

(iii) Fungal DNA extraction. A 50-ml volume of each ocular sample was frozenfor at least 1 h at 220°C. The DNA extraction was performed as described forthe standard fungi isolates. DNA was diluted in 10 ml 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 acnes PCR amplification with ocular samples were performed asdescribed by Hykin et al. (16).

(a) Detection of PCR inhibitors in ocular samples. The presence of PCRinhibitors in ocular fluids was tested before the study of clinical samples. To showthat vitreous or aqueous humor was not inhibitory to DNA extraction and PCR,50-ml samples of normal (not infected) vitreous and normal aqueous humor were

TABLE 1. Strains and source of ocular isolates analyzed by PCRamplification of rDNA

Straina

Predicted size (bp)of PCR product

with primers:

ITS1-ITS4

ITS86-ITS4

Alternaria alternata IOA-K3 570 292Aspergillus flavus CECT 2684 595 300Aspergillus fumigatus CECT 2071/IOA-K1 596 299Aspergillus niger CECT 2574/IOA-K2 599 300Aspergillus terreus CECT 2748 608 308Candida albicans CECT 1472 536 282Candida parapsilosis ATCC 22019/IOA-E1, IOA-E2 520 255Candida tropicalis CECT 1005 524 270Candida glabrata CECT 1448 820 360Candida kruseii ATCC 6258 510 294Fusarium oxysporum CECT 2154 544 283Fusarium solani CECT 2199 569 286Cryptococcus neoformans

var. neoformans SCNC SP1 556 320var. gatti SCNC C48 556 320

Scedosporium apiospermum HMM-K1 611 329

a IOA; ocular isolates obtained from the Instituto Oftalmologico de Alicante,Alicante, Spain; HMM, Ocular isolates obtained from Mostoles Hospital, Ma-drid, Spain; SCNC, Clinical isolates obtained from Spanish Cryptococcus neofor-mans Collection, Mycology Laboratory, Universidad Miguel Hernandez, Ali-cante, Spain; CECT, Spanish Type Culture Collection, Valencia, Spain; ATCC,Americans Type Culture Collection. Rockville, Md.

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spiked with 1 ml 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 purifiedusing the GeneClean II kit (Bio 101, Inc., Carlsbad, Calif.) as specified by themanufacturer and directly cycle sequenced in both directions using the BigDyeterminators Ready Reaction Kit (PE Applied Biosystems, Foster City, Calif.) onan ABI Prism automated DNA sequencer (model 377, version 2.1.1; AppliedBiosystems Warrington, United Kingdom). The primers used were ITS4 and ITS86.

(vi) Data analysis. The PCGENE program was used to ascertain the specificityof the method, including a large number of fungi. Ocular pathogenic fungi forwhich the rDNA sequence is available in GenBank were assayed for selectedprimers hybridization using this program. PCGENE facilitates the positive ornegative theoretical union of primers to the sequence target. After clustal align-ment of the selected sequences, fragment sizes were manually calculated. ITS2/5.8S rDNA sequences were analyzed by using the BLAST alignment program ofthe GenBank database (National Institutes of Health). The computer alignmentprovides a list of matching organisms, ranked in order of similarity between theunknown sequence and the sequence of the corresponding organism from thedatabase.

RESULTS

Standard fungal isolates. (i) PCR specificity. The primersused in this study (ITS1 and ITS4 for the first round amplifi-cation and ITS86 and ITS4 for the second round) successfullyamplified 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-ITS4 primer pairs with genomic DNA isolatedfrom 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 thespecificity of this method (Table 1). The sizes of the fragmentsobtained were in agreement with those obtained by PCR.

(ii) PCR sensitivity. The sensitivity was estimated using twokinds of samples: DNA dilutions (DNA was extracted from aculture and subsequently diluted) and culture dilutions (serialdilutions of cells were prepared and DNA extracted from eachculture).

For C. albicans DNA dilutions, the sensitivity of the firstPCR was found routinely (more than three times) to be 1 to 10fg (Fig. 2A). The seminested PCR, performed with 1 ml fromthe first PCR, was positive in all the DNA dilutions (Fig. 2B).The sensitivity for the mold A. fumigatus was found to be 10 to100 fg (Fig. 2C). The second round of PCR markedly improvedthis sensitivity to 1 fg, similar to the results obtained with C.albicans (Fig. 2D).

Using cell dilutions, the sensitivity of the PCR was routinelyfound to be 1 to 10 organisms (Fig. 3A and B). However, for A.fumigatus the sensitivity was lower; the first PCR was positiveonly when carried out with samples containing 10 to 102 or-ganisms, but with semnested PCR the sensitivity improved toless 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); C2: negativecontrol (double-distilled H2O [dd H2O]). (B) Seminested PCR prod-uct (ITS86-ITS4 primer pair). M, ladder marker pBR322 DNA/BsuRI(234 bp, white triangle; 213 bp; black triangle). The lanes are the sameas in panel A except as indicated. C2 (1st PCR); negative-controlsample from the first-round amplification; C2 (2nd PCR), negativecontrol (ddH2O).

FIG. 2. (A) Sensitivity of the first PCR (ITS1-ITS4 primer pair)with C. albicans genomic DNA. M, ladder marker GeneRuler 100bpDNA Ladder Plus (500 bp, black triangle); C2, negative control(ddH2O). (B) Sensitivity of the seminested PCR (ITS4-ITS86 primerpair) performed with 1 ml of the first-round product of C. albicansPCR. M, ladder marker pBR322 DNA/BsuRI (434 bp, white triangle;267 bp, black triangle); C2, negative control (ddH2O). (C) Sensitivityof the first PCR (ITS1-ITS4 primer pair) with A. fumigatus genomicDNA. M, ladder marker GeneRuler 100bp DNA Ladder Plus (500 bp,black triangle); C2, negative control (ddH2O). (D) Sensitivity of theseminested PCR (ITS4-ITS86 primer pair) performed with 1 ml of thefirst-round product of A. fumigatus PCR. M, ladder marker pBR322DNA/BsuRI (434 bp, white triangle; 267 bp, black triangle); C2,negative control (ddH2O).

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(iii) Detection of PCR inhibitors in ocular samples. Theexpected amplification products were obtained indicating thatno PCR inhibitors were present in the clinical samples (aque-ous humor and vitreous) after DNA extraction.

Ocular samples. Six cases of endophthalmitis and threecases of keratitis were analyzed by molecular and culture meth-ods. In the six cases of endophthalmitis, six aqueous samplesand two vitreous samples were taken. Table 2 shows the resultsof Gram’s stain, culture and PCR of all ocular samples. Sam-ples from patients 2, 5, and 8 were PCR negative with fungalprimers and positive with bacterial primers. The sample frompatient 2 was culture positive for coagulase-negative staphylo-cocci; the patient was successfully treated and responded wellto antibiotic therapy. The sample from patient 5 was PCRpositive with bacterial and P. acnes primers and culture posi-tive for P. acnes. The patient underwent anterior vitrectomywith intravitreal injection of antibiotics. Clinical and visualimprovement was rapid. Patient 8 is still under antibiotic treat-ment. The sample from patient 4 was negative for both PCRand culture analysis. The patient is under clinical observation,and the case is being reviewed every 3 months. The samplesfrom patients 3 and 7 were bacterial PCR negative and fungalPCR positive (Fig. 4). The sequence analysis and the cultureshowed a C. parapsilopsis infection. Patient 3 successfully fin-ished the antifungal treatment (fluconazole), and patient 7 isstill under fluconazole treatment. Corneal samples from pa-tients 1, 6, and 9 were positive with fungal primers (Fig. 4). Thesample from one of these patients (patient 1) was also positiveby culture, and fungi were detected in Gram’s stain; the samplefrom patient 9 was positive by culture and not detectable by

Gram’s stain; the sample from patient 6 was negative by bothtechniques (culture and Gram stain visualization) and a nestedPCR was necessary to detect the fungal DNA (Fig. 4). Patients1 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 wasbeing treated at Mostoles 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 identificationas S. apiospermum. Although DNA extraction from cornealsamples was not done, this case was also considered interestingfor the assessment of the technique.

Microscopic fungal visualization (Gram stains) was negativefor three of the six ocular samples (Table 2). Only the cornealsample from patient 6 and the aqueous sample from patient 8were 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 thePCR 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); C2, negative control (ddH2O). (B)Sensitivity of the seminested PCR (ITS4-ITS86 primer pair) per-formed with 1 ml of the first-round product of C. albicans PCR. M,ladder marker pBR322 DNA/BsuR1 (434 bp, white triangle; 267 bp,black triangle); C2, negative control (ddH2O). (C) Sensitivity of thefirst PCR (ITS1-ITS4 primer pair) with A. fumigatus cells. M, laddermarker GeneRuler 100bp DNA Ladder Plus (500 bp, black triangle);C2, negative control (ddH2O). (D) Sensitivity of the seminested PCR(ITS4-ITS86 primer pair) performed with 1 ml of the first-round prod-uct of A. fumigatus PCR. M, ladder marker pBR322 DNA/BsuRI (434bp, white triangle; 267 bp, black triangle); C2, negative control(ddH2O).

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-ITS4primer pair). M, Ladder marker pBR322 DNA/BsuRI (434 bp, whitetriangle; 267 bp, black triangle); P1, patient 1; P2, patient 2; P3, patient3; P4, patient 4; P5Aq, patient 5 aqueous sample; P5Vit, patient 5vitreous 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 DNAsequences obtained with the full-sequence ITS2 and partial-sequence 5.8S rDNA from the ocular samples demonstratedthat they were derived from the fungal ITS regions. Two ofthem were identical to the C. parapsilosis ITS2/5.8S rDNAregion, 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 identification 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 identification offungal pathogens based on amplification of ITS2/5.8S rDNAand molecular typing.

A good DNA extraction method is critical for PCR detectionto avoid the possibilities of false-negative results. We testedsome 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 notsatisfactory for all the tested fungal strains (A. niger or C.neoformans). Similar results with the use of other commercialkits, except for a QIAmp Tissue kit, for fungal DNA extractionhave been previously described (25). This protocol applied tovitreous ocular samples provides high-quality DNA that is de-void of PCR-inhibiting substances. The DNA loss is minimalbecause we have been able to amplify DNA from 1 to 10microorganisms (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 testedto date. The use of rRNA genes for identification of fungalspecies is based on the detection of conserved sequences in therDNA genes. Our results showed high fungal specificity withthe selected primers. All fungal strains were PCR positive, andall negative controls (human and bacterial DNA) were PCRnegative. This same protocol of extraction and amplification ofDNA from ocular samples was performed in an animal fungalinfection model (11). In this work, the fungal infection wasinduced 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 allinfection samples studied.

The primers used for PCR amplification were checked andfound to be specific for fungal rDNA; they did not targetprokaryotic rDNA sequences. Moreover, they targeted all therDNA sequences from ocular pathogenic fungi available indatabase.

As discussed above, the sensitivity for the first amplificationof the ITS/5.8S rDNA region was 1 fg for C. albicans and 10 fgfor A. fumigatus in water dilutions of DNA. Assuming a totalDNA content of 37 fg per organism (38), this amount is equiv-alent to less than one C. albicans cell. This is in agreement withthe fact that rRNA genes are multiple-copy genes, with 100 ormore copies within the fungal genome (32), making them idealtargets for PCR amplification and permitting the amplificationfrom 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 100organisms of A. fumigatus could be detected. As shown in Fig.2 and 3, the intensity of the band corresponding to the PCRcarried out with 10 to 100 fg of genomic C. albicans DNA issimilar to the intensity of the band corresponding to the PCRwith 1 to 10 microorganisms (the total DNA content is of 37 fgper organism) (38). For A. fumigatus, the intensity of the bandcorresponding to the PCR performed with 100 fg of DNA wassimilar to that obtained with 1 to 10 microorganisms (totalDNA of this mold could be estimated at approximately 35 Mb['100 fg]) (24). This means that there was no significant DNAloss during the DNA extraction. Nevertheless, to make sure, alarge number of experiments would be necessary because therange given for the DNA amount and the range given for cellnumbers in these preparations were not exactly the same.

Infectious agents were detected by PCR in eight of nine clinicalocular infections; five of them were positive by amplification withfungal primers, and three were positive by amplification withbacterial primers. In seven of the cases, the pathogen could alsobe retrieved by cultivation (two bacteria and five fungi). However,while the PCR result was obtained in a few hours, culturesneeded 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 shorterthan 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 keratitisa

Patient Sample Gram stain resultb Culture result Bacterial PCR/P. acnes PCR resultc Fungal PCR result/homology sequence

1 Corneal Scrape Positive Positive (2 days) ND Positive/A. fumigatus2 Aqueous ND* Positive (3 days) Positive/negative Negative3 Aqueous ND* Positive (6 days) Negative Positive/C. parapsilosis4 Aqueous ND* Negative Negative Negative5 Aqueous ND* Positive (13 days) Positive/positive Negative5 Vitreous Positive Positive (7 days) Positive/positive Negative6 Corneal scrape Negative Negative ND Positive/A. niger7 Aqueous ND* Negative Negative Negative7 Vitreous Negative Positive (7 days) Negative Positive/C. parapsilosis8 Aqueous ND* Negative Positive/negative Negative9 Corneal scrape Negative Positive (7 days) ND Positive/Alternaria alternata10 Culture of corneal scrape Positive Positive ND Positive/S. apiospermum

a ND, not done.b p, insufficient sample.c Where 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 bacterialorigin of the infection is complemented by the rapid identifi-cation of the fungus itself. For example, patient 1 had receivedintravitreal 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 tofluconazole (4, 19).

Analysis of sequences (5.8S/ITS region) from the databaseconfirmed that this method can be used to differentiate fungi atthe species level. Some studies show that fungal strains can bedistinguished on the basis of the size of the ITS/5.8S fragment(6, 41) and primary structural differences in the rDNA spacerregions (10, 46). However, although yeast demonstrated ahigher level of interspecies variability compared to other fungi,size determination based on agarose gel electrophoresis is notprecise enough to unmistakeably confirm the species identifi-cation. Table 1 shows that fragment sizes are very similar andtherefore very difficult to differentiate in an agarose gel. If thesize is determined by capillary electrophoresis (41), the timerequired 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 ofrestriction fragment length polymorphism analysis of ITS/5.8Sfragments, hybridization with a specific probe, and the specificnested PCR (10, 35, 46), could be useful to confirm a specificfungal infection, for example in endophthalmitis (frequentlyproduced by Candida, Aspergillus and Fusarium). However, therange of fungi capable of causing keratitis is significantly widerthan that of fungi capable of causing endophthalmitis. There-fore a large number of species causing infection could remainunidentified by these molecular methods. Specifically, in thecourse of this study, the identification for patients 9 and 10(Alternaria and Scedosporium) would be rather complicatedand time-consuming because of the need to consider the pos-sible involvement of these genera as pathogens. In contrast, theamplification and typing of the ITS region eliminates this re-quirement. In addition, the small size of the fragment permitsits sequencing in both directions at once, and the obtainedsequence gives enough information to identify the fungal species.

This method proved to be reproducible and very useful foreasy and rapid identification and classification of all the speciesincluded in the present work. This is the first time that theserDNA-specific primers have been successfully used for fungaldetection and identification in ocular samples. This PCR-basedmethod promises to be very effective for the diagnosis of fungalocular infections in the clinical setting. Compared with stan-dard laboratory techniques, it offers a significant reduction ofthe time required to establish the diagnosis. However, furtherstudies with a larger number of clinical samples are necessaryto assess the efficacy of the method.

ACKNOWLEDGMENTS

This work was supported by grant IMTEIA/1998/210 from theIMPIVA (Generalitat Valenciana, Spain) and a grant from InstitutoOftalmologico de Alicante (Alicante, Spain).

We thank Gema Salas, Stuart Ingham, and Maria Luz Campos(Facultad de Medicina, Universidad Miguel Hernandez, Alicante, Spain)for their technical assistance; Kathy Hernandez for her English lan-guage corrections; and Josefa Anton for scientific suggestions. We alsothank Elisa Amor from Mostoles Hospital (Madrid, Spain) for her col-laboration with the study of the Scedosporium apiospermum strain andfor providing information on the keratitis case associated with this strain.

REFERENCES

1. Accensi, F., J. Cano, L. Figuera, M. L. Abarca, and F. J. Cabanes. 1999. NewPCR methods to differentiate species in the Aspergillus niger aggregate.FEMS Microbiol. Lett. 180:191–196.

2. Affeldt, J. C., H. W. Jr. Flynn, R. K. Forster, S. Mandelbaum, J. G. Clarkson,and G. D. Jarus. 1987. Microbial endophthalmitis resulting from oculartrauma. Ophthalmology 94:407–413.

3. Alexandrakis, G., S. Jalali, and P. Gloor. 1998. Diagnosis of Fusariumkeratitis in an animal model using the polymerase chain reaction. Br. J.Ophthalmol. 82:306–311.

4. Borne, M. J., J. H. Elliot, and D. M. O’Day. 1993. Ocular fluconazoletreatment of Candida parapsilosis endophthalmitis after failed intravitrealamphotericin B. Arch. Ophthalmol. 111:1326–1327.

5. Carroll, N. M., E. E. Jaeger, S. Choudhury, A. A. Dunlop, M. M. Matheson,P. Adamson, N. Okhravi, and S. Lightman. 2000. Detection of and discrim-ination between gram-positive and gram-negative bacteria in intraocularsamples by using nested PCR. J. Clin. Microbiol. 38:1753–1757.

6. Chen, Y. C., J. D. Eisner, M. M. Kattar, S. L. Rassoulian-Barrett, K. LaFe,S. L. Yarfitz, A. P. Limaye, and B. T. Cookson. 2000. Identification ofmedically important yeasts using PCR-based detection of DNA sequencepolymorphisms in the internal transcribed spacer 2 region of the rRNAgenes. J. Clin. Microbiol. 38:2302–2310.

7. Driebe, W. T., S. Mandelbaum, R. K. Forster, L. K. Schwartz, and W. W.Culbertson. 1986. Pseudophakic endophthalmitis. Diagnosis and manage-ment. Ophthalmology 93:442–448.

8. Dunlop, A. A., E. D. Wright, S. A. Howlader, I. Nazrul, R. Husain, K.McClellan, and F. A. Billson. 1994. Suppurative corneal ulceration in Ban-gladesh. A study of 142 cases examining the microbiological diagnosis, clin-ical and epidemiological features of bacterial and fungal keratitis. Aust.N. Z. J. Ophthalmol. 22:105–110.

9. Einsele, H., H. Hebart, G. Roller, J. Loffler, I. Rothenhofer, C. A. Muller,R. A. Bowden, J. van Burik, D. Engelhard, L. Kanz, and U. Schumacher.1997. Detection and identification of fungal pathogens in blood by usingmolecular probes. J. Clin. Microbiol. 35:1353–1360.

10. Esteve-Zarzoso, B., C. Belloch, F. Uruburu, and A. Querol. 1999. Identifi-cation of yeast by RFLP analysis of the 5.8S rRNA gene and the tworibosomal internal transcribed spacers. Int. J. Syst. Bacteriol. 49:329–337.

11. Ferrer, C., S. Frases, F. Colom, J. L. Alio, J. L. Abad, and M. E. Mulet. 2000.Molecular diagnosis of fungal ocular infections in an animal model. Rev.Iberoam. Micol. 17:S134.

12. Forster, R. K., R. L. Abbott, and H. Gelender. 1980. Management of infec-tious endophthalmitis. Ophthalmology 87:313–319.

13. Han, D. P., S. R. Wisniewski, L. A. Wilson, M. Barza, A. K. Vine, B. H. Doft,and S. F. Kelsey. 1996. Spectrum and susceptibilities of microbiologic iso-lates in the endophthalmitis vitrectomy study. Am. J. Ophthalmol. 122:1–17.

14. Haynes, K. A., T. J. Westerneng, J. W. Fell, and W. Moens. 1995. Rapiddetection and identification of pathogenic fungi by polymerase chain reac-tion amplification of large subunit ribosomal DNA. J. Med. Vet. Mycol.33:319–325.

15. Hidalgo, J. A., G. J. Alangaden, D. Eliott, R. A. Akins, J. Puklin, G. Abrams,and J. A. Vazquez. 2000. Fungal endophthalmitis diagnosis by detection ofCandida albicans DNA in intraocular fluid by use of a species-specific poly-merase chain reaction assay. J. Infect. Dis. 181:1198–1201.

16. Hykin, P. G., K. Tobal, G. McIntyre, M. H. Matheson, H. M. Towler, andS. L. Lightman. 1994. The diagnosis of delayed post-operative endoph-thalmitis by polymerase chain reaction of bacterial DNA in vitreous samples.J. Med. Microbiol. 40:408–415.

17. Jaeger, E. E., N. Carroll, S. Choudhury, A. A. Dunlop, H. M. Towlern, M. M.Matheson, P. Adamson, N. Okhravi and S. Lightman. 2000. Rapid detectionand identification of Candida, Aspergillus, and Fusarium species in ocularsamples using nested PCR. J. Clin. Microbiol. 38:2902–2908

18. Jordan, J. A. 1994. PCR identification of four medically important Candidaspecies by using a single primer pair. J. Clin. Microbiol. 32:2962–2967.

19. Kauffman, C. A., S. F. Bradley, and A. K. Vine. 1993. Candida endophthalmi-tis associated with intraocular lens implantation: efficacy of fluconazole ther-apy. Mycoses 36:13–17.

20. Knox, C. M., V. Cevellos, and D. Dean. 1998. 16S ribosomal typing foridentification of pathogens in patients with bacterial keratitis. J. Clin. Mi-crobiol. 36:3492–3496.

21. Knox, C. M., V. Cevellos, T. P. Margolis, and D. Dean. 1999. Identificationof bacterial pathogens in patients with endophthalmitis by 16S ribosomalDNA typing. Am. J. Ophthalmol. 128:511–512.

22. Kunimoto, D. Y., T. Das, S. Sharma, S. Jalali, A. B. Majji, U. Gopinathan,S. Athmanathan, and T. N. Rao. 1999. Microbiologic spectrum and suscep-tibility of isolates: part 1 postoperative endophthalmitis. EndophthalmitisResearch Group. Am. J. Ophthalmol. 128:240–242.

23. Kunimoto, D. Y., T. Das, S. Sharma, S. Jalali, A. B. Majji, U. Gopinathan,S. Athmanathan, and T. N. Rao. 1999. Microbiologic spectrum and suscep-tibility of isolates II. Posttraumatic endophthalmitis. Endophthalmitis Re-search Group. Am. J. Ophthalmol. 128:242–244.

24. Kupfer, D. M., C. A. Reece, S. W. Clifton, B. A. Roe, and R. A. Prade. 1997.

2878 FERRER ET AL. J. CLIN. MICROBIOL.

on February 14, 2020 by guest

http://jcm.asm

.org/D

ownloaded from

Multicellular ascomycetous fungal genome contain more than 8000 genes.Fungal Genet. Biol. 31:364–372.

25. Loeffler, J., H. Hebart, U. Shumacher, H. Reitze, and H. Einsele. 1997.Comparison of different methods for extraction of DNA of fungal pathogensfrom cultures and blood. J. Clin. Microbiol. 35:3311–3312.

26. Lohmann, C. P., M. Heeb, H. J. Linde, V. P. Gabel, and U. Reischl. 1998.Diagnosis of infectious endophthalmitis after cataract surgery by polymerasechain reaction. J. Cataract Refractive Surg. 24:821–826.

27. Lohmann, C. P., H. J. Linde, and U. Reischl. 2000. Improved detection ofmicroorganisms by polymerase chain reaction in delayed endophthalmitisafter cataract surgery. Ophthalmology 107:1047–1051.

28. Lopez-Llorca, L. V. and T. Carbonell. 1999. Use of almond mesocarp forproduction of the entomopathogenic fungus verticillium lecanii. Can. J. Mi-crobiol. 44:886–895.

29. Lott, T. J., B. M. Burns, R. Zancope-Oliveira, C. M. Elie, and E. Reiss. 1998.Sequence analysis of the internal transcribed spacer 2 (ITS2) from yeastspecies within the genus Candida. Curr. Microbiol. 36:63–9.

30. Lott, T. J., R. J. Kuykendall, and E. Reiss. 1993. Nucleotide sequenceanalysis of the 5.8S rDNA and adjacent ITS2 region of Candida albicans andrelated species. Yeast 9:1199–1206.

31. Makimura, K., S. Y. Murayama, and H. Yamaguchi. 1994. Detection of awide range of medically important fungi by the polymerase chain reaction. J.Med. Microbiol. 33:358–364.

32. Maleszka, R., and G. D. Clark-Walker. 1993. Yeasts have a four-fold vari-ation in ribosomal DNA copy number. Yeast 9:53–58.

33. Miyakawa, Y., T. Mabuchi, K. Kahaya, and Y. Fukazawa. 1992. Isolation andcharacterization of a species-specific DNA fragment for detection of Can-dida albicans by polymerase chain reaction. J. Clin. Microbiol. 30:894–900.

34. Okhravi, N., P. Adamson, N. Carroll, A. Dunlop, M. M. Matheson, H. M.Towler, and S. Lightman. 2000. PCR-based evidence of bacterial involve-ment in eyes with suspected intraocular infection. Investig. Ophthalmol.Visual Sci. 41:3474–3479.

35. Okhravi, N., P. Adamson, R. Mant, M. M. Matheson, G. Midgley, H. M.Towler, and S. Lightman. 1998. Polymerase chain reaction and restrictionfragment length polymorphism mediated detection and speciation of Can-dida spp. causing intraocular infection. Investig. Ophthalmol. Visual Sci.39:859–866.

36. Okhravi, N., P. Adamson, M. M. Matheson, H. M. Towler, and S. Lightman.

2000. PCR-RFLP-mediated detection and speciation of bacterial speciescausing endophthalmitis. Investig. Ophthalmol. Visual Sci. 41:1438–1447.

37. Reiss, E., K. Tanaka, G. Bruker, V. Chazalet, D. Coleman, J. P. Debeaupuis,R. Hanazawa, J. P. Latge, J. Lortholary, K. Makimura, C. J. Morrison, S. Y.Murayama, S. Naoe, S. Paris, J. Sarfati, K. Shibuya, D. Sullivan, K. Uchida,H. Yamaguchi. 1998. Molecular diagnosis and epidemiology of fungal infec-tions. Med. Mycol. 36:249–257.

38. Riggsby, W. S., L. J. Torres-Bauza, J. W. Wills, and T. M. Townes. 1982.DNA content, kinetic complexity, and the ploidy question in Candida albi-cans. Mol. Cell. Biol. 2:853–862.

39. Tang, C. M., D. W. Holden, A. Aufauvre-Brown, and J. Cohen. 1993. Thedetection of Aspergillus spp. by the polymerase chain reaction and its eval-uation in bronchoalveolar lavage fluid. Am. J. Respir. Dis. 148:1313–1317.

40. Therese, K. L., A. R. Anand, and H. N. Madhavan. 1998. PCR in thediagnosis of bacterial endophthalmitis. Br. J. Ophthalmol. 82:1078–1082.

41. Turenne, C. Y., S. E. Sanche, D. J. Hoban, J. A. Karlowsky, and A. M.Kabani. 1999. Rapid identification of fungi by using the ITS2 genetic regionand an automated fluorescent capillary electrophoresis system. J. Clin. Mi-crobiol. 37:1846–1851.

42. Upadhyay, M. P., P. C. Karmacharya, S. Koirala, N. R. Tuladhar, L. E.Bryan, G. Smolin, and J. P. Whitcher. 1991. Epidemiologic characteristics,predisposing factors, and etiologic diagnosis of corneal ulceration in Nepal.Am. J. Ophthalmol. 111:92–99.

43. Van Burik, J. A. H., R. W. Schreckhise, T. C. White, and R. A. Bowden. 1998.Comparison of six extraction techniques for isolation of DNA from filamen-tous fungi. Med. Mycol. 36:299–303.

44. White, T. J., T. Bruns, S. Lee, and S. Tailor. 1990. Amplification and directsequencing of fungal ribosomal RNA genes for phylogenetics, p. 315–322. InM. A. Innins, D. H. Gelfand, J. J. Sninsky, and T. J. White (ed.), PCRprotocols. A guide to methods and applications. Academic Press, Inc., SanDiego, Calif.

45. Wiedbrauk, D. L., J. C. Werner, and A. M. Drevon. 1995. Inhibition of PCRby aqueous and vitreous fluids. J. Clin. Microbiol. 33:2643–2646.

46. Williams, D. W., M. J. Wilson, M. A. O. Lewis, and A. J. C. Potts. 1995.Identification of Candida species by PCR and restriction fragment lengthpolymorphism analysis of intergenic spacer regions of ribosomal DNA. J.Clin. Microbiol. 33:2476–2479.

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