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Rapid Identification of Dimorphic and Yeast Like Fungal Pathogens Using Specific DNA Probes

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Rapid Identification of Dimorphic and Yeast-Like Fungal

Pathogens Using Specific DNA Probes

MARK D. LINDSLEY,* STEVEN F. HURST, NAUREEN J. IQBAL,ANDCHRISTINE J. MORRISON Mycotic Diseases Branch, Division of Bacterial and Mycotic Diseases, Centers for

Disease Control and Prevention, Atlanta, Georgia

Received 31 May 2001/Returned for modification 22 July 2001/Accepted 7 August 2001

Specific oligonucleotide probes were developed to identify medically important fungi that display yeast-like morphology in vivo. Universal fungal primers ITS1 and ITS4, directed to the conserved regions of ribosomal DNA, were used to amplify DNA fromHistoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis,

Paracoccidioides brasiliensis, Penicillium marneffei, Sporothrix schenckii, Cryptococcus neoformans, fiveCandida

species, andPneumocystis carinii. Specific oligonucleotide probes to identify these fungi, as well as a probe to detect all dimorphic, systemic pathogens, were developed. PCR amplicons were detected colorimetrically in an enzyme immunoassay format. The dimorphic probe hybridized with DNA fromH.capsulatum,B.dermatitidis,

C.immitis,P.brasiliensis, andP.marneffeibut not with DNA from nondimorphic fungi. Specific probes forH.

capsulatum,B.dermatitidis, C. immitis,P. brasiliensis,P. marneffei, S. schenckii,C. neoformans, andP.carinii

hybridized with homologous but not heterologous DNA. Minor cross-reactivity was observed for the B.

dermititidisprobe used againstC.immitisDNA and for theH.capsulatumprobe used againstCandida albicans

DNA. However, theC.immitisprobe did not cross-react withB.dermititidisDNA, nor did the dimorphic probe hybridize withC.albicansDNA. Therefore, these fungi could be differentiated by a process of elimination. In conclusion, probes developed to yeast-like pathogens were found to be highly specific and should prove to be useful in differentiating these organisms in the clinical setting.

The incidence of disease caused by pathogenic and oppor-tunistic fungi has been increasing over the past decade (1, 2, 4, 25, 34). Such increases are primarily the result of the human immunodeficiency virus epidemic and advances in modern medicine that maintain or prolong the lives of severely ill patients (12, 25, 34). Moreover, the true burden of fungal disease is most certainly underestimated because of the insen-sitivity of present diagnostic methods (8, 18, 25). Diagnosis of fungal infections is typically made by isolation of the infecting organism in culture, by serologic assays, or through histopatho-logic examination of tissue (13, 27). Histopathohistopatho-logic diagnosis is advantageous because it is more rapid than culture. Patho-genic fungi may require 2 to 3 weeks or longer to grow (39, 40). A positive culture may also represent colonization rather than true invasion, especially when opportunistic organisms are iso-lated (7, 16). Furthermore, an infectious etiology may not be suspected at the time of biopsy and the tissue is often placed in fixative, making culture impossible. Histopathologic diagnosis can also be more rapid than serology. Serologic tests on a sin-gle serum sample to detect circulating antifungal antibodies may be inconclusive (especially in immunosuppressed pa-tients). The acquisition of paired acute- and convalescent-phase sera, which is necessary for definitive serologic diagnosis, requires an additional 3 to 4 weeks before convalescent-phase serum can be obtained (27). Therefore, histopathologic exam-ination of tissue sections may be the most rapid or only way in which to diagnose invasive fungal disease.

Histopathologic diagnosis of fungal infections is typically made through morphological criteria using reagents that

pref-erentially stain fungal structures (6). However, fungi may pre-sent with atypical morphological features, making definitive histopathologic identification difficult (19). Other methods for diagnosis are then required. Presently, the immunofluores-cence assay (either direct or indirect) using a specific antibody is the primary method available for in situ identification (18, 31). However, polyclonal antibodies to detect specific fungi are in limited supply and are generally not available commercially. Generating specific polyclonal antibodies to replace decreasing supplies is time consuming, and the replacement antibodies are often not of the same avidity or specificity as the original. Monoclonal antibody preparations can provide a reproducible supply of reagent that is usually highly specific but may be less sensitive than the corresponding polyclonal antibodies (8). The relatively recent development of automated DNA synthesis has allowed production of molecular probes with consistently defined properties that may result in increased test sensitivity, specificity, and reproducibility.

Past research in the molecular identification of fungi has typically concentrated on a single species or genus of fungus (3, 9, 14, 21, 22, 26, 29, 30, 32, 33, 37) or has used methods that may be cumbersome for the routine diagnostic laboratory to perform (35, 36, 38). The present study employed a modifica-tion of a PCR-enzyme immunoassay (PCR-EIA) method (10, 11) for amplification and differentiation of the major fungal pathogens that possess a yeast-like morphology in vivo. This method allows amplification and specific identification of DNA from all fungi, using a convenient method that requires little specialized equipment. Fungal DNA is amplified using univer-sal fungal primers (ITS1 and ITS4) directed towards the rRNA gene. The rRNA gene is a common target for molecular iden-tification of fungi because this area of the genome contains both unique and conserved regions (41). PCR amplification * Corresponding author. Mailing address: Mailstop G-11, CDC,

1600 Clifton Rd., NE, Atlanta, GA 30333. Phone: (404) 639-4340. Fax: (404) 639-3546. E-mail: mil6@cdc.gov.

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using the ITS1 and ITS4 primers produces an approximately 600-bp amplicon which contains conserved regions among fungi, including the sequence for ITS3 (41). The hybridization EIA then colorimetrically detects amplicons using biotinylated ITS3 and specific oligonucleotide probes directed to rDNA regions unique for each fungus. The combination of universal fungal PCR amplification and colorimetric detection creates a PCR-EIA that can potentially detect and specifically identify DNA from any fungus. Whereas the ultimate goal of this re-search is to identify fungi in tissue, the specificity of these probes was first tested using the PCR-EIA format and DNA isolated from cultured fungal isolates. This paper describes the development of specific oligonucleotide probes to identify fun-gal pathogens that possess yeast-like morphology in vivo.

MATERIALS AND METHODS

DNA isolation.One loopful of yeast-phase Blastomyces dermatitidis(strain

4478, KL-1 [ATCC 26198], or A2 [ATCC 60916]), was inoculated into 10 ml of brain heart infusion broth (BD, Sparks, Md.) in a 50-␮l Erlenmeyer flask and was incubated at 37°C on a rotary shaker (140 rpm) for 48 to 72 h. The suspension was then transferred to a 30-ml Oak Ridge centrifuge tube (Nalge, Rochester,

N.Y.) and was centrifuged for 3 min at 2,000⫻g. Genomic DNA was extracted

and purified using a commercial kit (PureGene Yeast and Gram Positive DNA Isolation Kit; Gentra Systems Inc., Minneapolis, Minn.) following the manufac-turer’s protocol.

Mold-phase cultures ofSporothrix schenckii(ATCC 58251) andPenicillium

marneffei(strains ATCC 64101, ATCC 58950, and JH05 [gift of William Merz, Johns Hopkins Medical School, Baltimore, Md.]) were grown in 50 ml of Sab-ouraud dextrose broth (Difco) in 250-ml Erlenmeyer flasks and were incubated at 25°C on a rotary shaker for 5 days. Growth was harvested by vacuum filtration through sterile filter paper, and the cellular mat was washed three times with

sterile, distilled H2O by filtration. The cellular mat was then removed from the

filter and placed into a sterile petri plate which was then sealed around the edges

with Parafilm (American Can, Neenah, Wis.) and was frozen at⫺20°C until

used.

DNA was extracted by grinding the cellular mats with a mortar and pestle in the presence of liquid nitrogen. Just before use, a portion of the frozen cellular mat, approximately equal in size to a quarter, was removed from the petri plate with sterile forceps and was placed into an ice-cold, sterile mortar (diameter, 6 in.). Liquid nitrogen was added to cover the mat and was added as needed to keep the mat frozen during grinding. The fungal mat was ground into a fine powder with a sterile pestle. Fungal DNA was then extracted and purified using serial proteinase K and RNase treatments followed by phenol extraction and ethanol precipitation by conventional methods (24).

Other DNA was kindly provided as a gift from the following persons:

His-toplasma capsulatum(strains G186B [ATCC 26030], Down’s, FLs-1, and B293

[var. duboisii]), Brent Lasker, Centers for Disease Control and Prevention

(CDC), Atlanta, Ga.;Coccidioides immitis(strains C635 and C735), Garry Cole,

Medical College of Ohio, Toledo, Ohio;Paracoccidioides brasiliensis(strains 265,

Pb18, rh, and soil [soil isolate from Venezuela]), Maria Jose Soares Mendes Giannini, Faculdade de Ciencias Farmaceuticas, Universidade Estadual Paulista, Araraquara, Brazil, and Juan McEwen, Corporacion Para Investigaciones

Bio-logicas, Medellin, Colombia;Cryptococcus neoformans(strains 9759-MU-1

[se-rotype A], BIH409 [se[se-rotype B], K24066TAN [se[se-rotype C], and 9375 [se[se-rotype

D]) and allCandidaspecies DNA (Candida albicans[strain B311],Candida

glabrata[CDC Y-65],Candida krusei[CDC 259-75],Candida tropicalis[CDC 38], andCandida parapsilosis[ATCC 22019]), Cheryl Elie, CDC; andPneumocystis carinii(rat isolate), Charles Beard, CDC.

Primers and probes.All primers and probes were synthesized by␤-cyanoethyl

phosphoramidite chemistry using a 394 or expedite automated DNA synthesizer (PE Applied Biosystems, Foster City, Calif.). ITS3, a universal fungal sequence located in the 5.8S region of the rRNA gene and contained within the region

amplified by ITS1 and ITS4 primers (23, 41), was biotinylated at the 5⬘end by

incorporating dimethyoxytrityl-biotin-carbon-6-phosphoramidite during its syn-thesis. This biotinylated probe (ITS3-B) was then purified by reverse-phase liquid chromatography. Digoxigenin-labeled probes were synthesized with a

5⬘-termi-nal amine group using 5⬘Amino-Modifier C6 (Glen Research, Sterling, Va.),

mixed with a 10-fold-molar excess of digoxigenin-3-O-methylcarbonyl-ε

-amino-caproic acidN-hydroxysuccinimide ester (Roche Molecular Biochemicals,

Indi-anapolis, Ind.) in 0.1 M sodium carbonate buffer, pH 9.0, and incubated at ambient temperature overnight. The digoxigenin-labeled probes were then pu-rified by reverse-phase high-pressure liquid chromatography (5). Sequences and locations in the rRNA gene of these primers and probes are depicted in Table 1 and Fig. 1, respectively. All primers and probes were synthesized by the CDC Biotechnology Core Facility.

Microbe-specific probes.DNA sequences of the ITS2 region of the fungal

rRNA gene were obtained from GenBank (Table 1). Those fungi that did not

have sequences available in GenBank (P.brasiliensis,S.schenckii, andP.

marnef-TABLE 1. Sequences of oligonucleotide primers and probes

Oligonucleotide probe

or primer Sequence (5⬘to 3⬘) Source orreference Oligonucleotide labeling

PCR primers

ITS1 TCCGTAGGTGAACCTGCGG 31 Universal forward primer

ITS4 TCCTCCGCTTATTGATATGC 31 Universal reverse primer

Probes

ITS3-B GCATCGATGAAGAACGCAGC 31 5⬘-Biotin-labeled universal capture probe

Dm GGACGTGCCCGAAATGCAGTGGCGG U18363a 5-Digoxigenin-labeled probe for all endemic dimorphic fungi Hc ACCATCTCAACCTCCTTTTTCACACCAGG U18363 5⬘-Digoxigenin-labeled probe forH. capsulatum

Bd GGTCTTCGGGCCGGTCTCCCC U18364 5⬘-Digoxigenin-labeled probe forB. dermatitidis

Ci CTCTTTTTTTTATTATATCC U18360 5⬘-Digoxigenin-labeled probe forC. immitis

Pb CACTCATGGACCCCGG AF322389 5⬘-Digoxigenin-labeled probe forP. brasiliensis

Ss GACGCGCAGCTCTTTTTA AF117945 5⬘-Digoxigenin-labeled probe forS. schenckii

Pm GGGTTGGTCACCACCATA L37406 5⬘-Digoxigenin-labeled probe forP. marneffei

Cn CCTATGGGGTAGTCTTCGG L14068 5⬘-Digoxigenin-labeled probe forC. neoformans

Pc GTAGTAGGGTTAATTCAATT L27658 5⬘-Digoxigenin-labeled probe forP. carinii

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aGenBank sequence accession number.

FIG. 1. Diagram of hybridization sites of primers and probes. Hy-bridization sites for the ITS1 and ITS4 primers are in the phyloge-netically conserved 18S and 28S rDNA regions, and arrows desig-nate the direction of amplification (ITS1, forward primer; ITS4, reverse primer). ITS3 (biotin) represents the biotinylated, universal fungal probe which binds in the phylogenetically conserved, 5.8S rDNA region. Probe (Digox.) represents digoxigenin-labeled, microbe-specific probes which bind to the less highly conserved ITS2 region.

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fei) were sequenced using a dye terminator cycle sequencing kit (ABI PRISM; Applied Biosystems, Perkin-Elmer, Foster City, Calif.), and sequences have since been deposited with GenBank by our laboratory or by others (accession numbers:

S.schenckii, AF117945;P.brasiliensis, AF322389; andP.marneffei, L37406). Briefly, primary DNA amplifications were conducted using ITS1 and ITS4 as primers. The DNA was purified using QIAquick Spin Columns (Qiagen Corp.,

Chats-worth, Calif.) and was eluted with 50␮l of heat-sterilized Tris-EDTA buffer

(10 mM Tris, 1 mM EDTA, pH 8.0). Sequencing was performed in both the

for-ward and reverse directions. The reaction mix (20␮l) containing 9.5 ␮l of

terminator premix, 2␮l (1 ng) of DNA template, 1␮l of primer (either a forward

or reverse primer, 3.2 pmol), and 7.5␮l of heat-sterilized, distilled H2O was

placed into a preheated (96°C) Perkin-Elmer 9600 thermal cycler for 25 cycles of 96°C for 10 s, 50°C for 5 s, and 60°C for 4 min. The PCR product was then purified before sequencing using CentriSep spin columns (Princeton

Separa-tions, Inc., Adelphia, N.J.). DNA was then vacuum dried, resuspended in 6␮l of

formamide-EDTA (5␮l deionized formamide plus 1␮l of 50 mM EDTA, pH

8.0), and denatured for 2 min at 90°C before subjection to sequencing using an automated capillary DNA sequencer (model 373; ABI Systems, Bethesda, Md.). Sequences were aligned and a comparison was performed to determine unique sequences that could be used for the development of specific digoxigenin-labeled oligonucleotide probes. The initial screen for specificity of the probe sequences was performed using basic local alignment search tool (BLAST) software (GCG, Madison, Wis.). Probe sequences determined to be unique were then synthesized and digoxigenin labeled as described above.

PCR conditions.The PCR mix consisted of 10 mM Tris-HCl buffer containing

50 mM KCl, pH 8.0 (Roche), 1.5 mM MgCl2(Roche), 0.2 mM deoxynucleoside

triphosphate (TaKaRa Shuzo Co. Ltd., Otsu, Shiga, Japan), and 1.25 U ofTaq

polymerase (TaKaRa Shuzo). Primers ITS1 and ITS4 were added to a final concentration of 0.2 mM each. Template DNA was added at a final

concentra-tion of 1 ng per 50␮l of reaction mix. For each experiment, at least one reaction

tube received water in place of template DNA as a negative control. Amplifica-tion was performed in a Model 9600 thermocycler (Perkin-Elmer, Emeryville, Calif.). Initial denaturation of template DNA was achieved by heating at 95°C for 5 min. This was followed by 30 cycles of 30 s at 95°C, 30 s at 58°C, and 1 min at 72°C. A final extension step was conducted for 10 min at 72°C. Appropriate controls were included and PCR contamination precautions were followed (11, 20).

Agarose gel electrophoresis.Successful PCR amplification was confirmed by

visualization of PCR amplicons after agarose gel electrophoresis. Gels consisted of 1% agarose LE (Boehringer Mannheim, Indianapolis, Ind.) and 1% NuSieve GTG agarose (FMC Bioproducts, Rockland, Maine) or 2% Metaphore agar (FMC Bioproducts) dissolved in Tris-borate-EDTA buffer (0.1 M Tris, 0.09 M boric acid, 0.001 M EDTA, pH 8.4). Five microliters of the PCR amplicons was

combined with 1␮l of tracking dye (Roche) and was then added to each well of

the agarose gel. Electrophoresis was conducted at 70 to 80 V for 45 to 60 min. The gel was stained with ethidium bromide for 30 min and washed in deionized water for 30 min before examination on a UV transilluminator.

EIA.EIA identification of PCR products was performed as previously

de-scribed (10, 11), with minor modifications (Fig. 2). Briefly, tubes containing 10␮l

of heat-denatured (5 min at 95°C) PCR amplicons were placed on ice, and 200

␮l of hybridization buffer (4⫻SSC [1⫻SSC is 0.15 M NaCl plus 0.015 M sodium

citrate], pH 7.0, 0.02 M HEPES, 0.002 M EDTA, and 0.15% Tween 20)

con-taining 10 ng of ITS3-B and 10 ng of a digoxigenin-labeled specific probe was added. Samples were mixed and incubated at 37°C for 1 h. One hundred micro-liters of the mixture was added in duplicate to wells of a strepavidin-coated, 96-well, microtiter plate (Roche) and was incubated at ambient temperature for 1 h on a microtiter plate shaker (⬃350 rpm; Labline Instruments, Melrose Park, Ill.). Microtiter plates were washed six times with 0.01 M phosphate-buffered saline, pH 7.2 (GibcoBRL, Life Technologies, Grand Island, N.Y.), containing 0.05% Tween 20 (Sigma Chemical Co., St. Louis, Mo.) (PBST) before addition

of 100␮l of a 1:1,000 dilution of horseradish peroxidase-labeled, anti-digoxigenin

antibody (150 U/ml; Roche) per well. Plate contents were incubated for 1 h at ambient temperature with shaking and were then washed six times with PBST.

3,3⬘,5,5⬘-Tetramethylbenzidine (TMB)-H2O2 substrate (Kirkegaard & Perry,

Gaithersburg, Md.) was then added to the wells, and the color reaction was allowed to develop at ambient temperature for 15 min. The optical density of each well was immediately read at a wavelength of 650 nm in a UVMax micro-titer plate reader (Molecular Devices, Sunnyvale, Calif.). The optical density of the duplicate wells was averaged and used in the analysis of the results. The optical density results were then converted to an EIA index (EI), which was calculated by dividing the optical density of the wells which had received test DNA by the optical density of the PCR water control as follows: optical density

of test DNA/optical density of water blank⫽EI.

Statistical analysis.Student’sttest was used to determine differences between

the mean EIs of probe hybridization to homologous DNA and those of probe hybridization to heterologous DNA. Differences were considered significant

whenPwas less than or equal to 0.05.

RESULTS

Confirmation of DNA amplification.To verify that the spe-cific DNA target was appropriately amplified and was of the expected size, the PCR amplicons were subjected to agarose gel electrophoresis and bands were visualized after ethidium bromide staining. The amplification of the rRNA gene using the ITS1 and ITS4 primers resulted in an approximately 600-bp-long amplicon for all fungi tested. The molecular sizes of amplicons were especially similar among the systemic, dimor-phic fungi (Fig. 3). The greatest differences in amplicon size were observed among the fiveCandidaspecies tested and were particularly pronounced forC.glabrataandC.kruseicompared to all otherCandida species (Fig. 3). However, specific iden-tification of the fungi using amplicon size alone was not possi-ble and is not generally recommended (28). Therefore, probes were designed to specifically identify each fungus using a mod-ification of an EIA method previously described (10, 11).

Probe specificity.Digoxigenin-labeled probes directed to the ITS2 region of rDNA were designed to specifically detect PCR amplicons from the most medically important yeast-like fungi. In addition to the microbe-specific probes, a probe was also designed as a primary screening probe with which to identify only the systemic, dimorphic fungal pathogens. The specificity of these probes was confirmed using the PCR-EIA method in a checkerboard pattern (Table 2). The dimorphic screening probe (Dm) successfully hybridized with PCR amplicons from all strains of the major systemic, dimorphic fungi tested (H. capsulatum, B. dermatitidis, C. immitis, P. brasiliensis, and P. marneffei) but not with DNA from any strain of the other yeast-like fungi (S.schenckii,C.neoformans,Candidaspecies, andP.carinii).

[image:3.587.46.280.74.165.2]

Microbe-specific probes, designed to detect only DNA am-plified from their homologous fungus, were tested against PCR amplicons from all strains of both homologous as well as het-erologous yeast-like fungi. The results in Table 2 demonstrate that the microbe-specific probes hybridized with DNA from homologous fungi and not with DNA from heterologous fungi (P ⬍ 0.001 or P ⬍ 0.05) with minor exceptions. There was FIG. 2. Diagram of PCR-EIA procedure. PCR product is heat

de-natured and incubated with both a biotinylated capture probe (ITS3-B) and a microbe-specific digoxigenin-labeled probe (Dig-Probe) before addition to the wells of a streptavidin-coated (AAAAAA) microtiter plate. Probe hybridization is then detected using a peroxidase-labeled (P), anti-digoxigenin antibody (Ab) and a colorimetric substrate-hy-drogen peroxide mixture (TMB-H2O2). Specifics are described in

Ma-terials and Methods.

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some reactivity of theB.dermatitidisprobe observed when it was tested againstC.immitisDNA. However, the hybridization signal for theB.dermatitidisprobe tested againstB.dermatitidis DNA was statistically greater than forC.immitisDNA (11.9⫾ 2.0 versus 4.3⫾0.8;P⬍0.01). In addition, the reverse (i.e., the C.immitisprobe tested againstB.dermititidisDNA) was neg-ative and could be used to differentiate the two fungi by a process of elimination. There was also a hybridization signal observed for theH.capsulatumprobe reacted with DNA from C. albicans(15.8⫾1.4 versus 6.5 ⫾1.0;P⬍ 0.001), but no signal was observed for any of the otherCandidaspecies tested using this probe. The dimorphic probe, however, did not hy-bridize withC.albicansDNA, and theC.albicansprobe did not hybridize withH.capsulatumDNA (10, 11; data not shown). Therefore, analyzing results obtained using these probes would eliminate any doubt regarding the identity of the organism from which the DNA was derived.

Confirmation of probe specificity using multiple strains of homologous and heterologous fungi.To further analyze each probe’s capacity to hybridize with only DNA from homologous fungi, DNA from multiple strains of each of the systemic, di-morphic fungi was tested in the PCR-EIA (Table 3). The probe designed to identify all systemic, dimorphic fungi hybridized with DNA from all strains ofH. capsulatum, B. dermatitidis, C. immitis, andP.brasiliensis tested. In addition, the probes specific for individual dimorphic fungi hybridized only to DNA isolated from homologous fungi but not to DNA isolated from heterologous fungi (Table 3). The minor hybridization signal observed for theB.dermatitidisprobe tested againstC.immitis DNA was similar for both strains ofC.immitistested.

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Sensitivity of probes using PCR-EIA.To assess the limit of sensitivity of the PCR-EIA method, compared to that for de-tection of amplicons by agarose gel electrophoresis,H. capsu-latum(Down’s strain) DNA was serially diluted prior to PCR FIG. 3. Agarose gel of amplified products from yeast-like fungi. Lane abbreviations (left to right): MW, molecular weight markers (HaeIII digest of⌽X174 plasmid; Roche);H.capsulatum, DNA amplified fromH.capsulatumstrains B293, Down’s, and Fls-1;B.dermatitidis, DNA amplified fromB.dermatitidisstrains 4478, KL-1, and A2;C.immitis, DNA amplified fromC.immitisstrains C635 and C735;C.neoformans, DNA amplified fromC.neoformansstrains 9759-MU-1 (serotype A), BIH409 (serotype B), K24066TAN (serotype C), and 9375 (serotype D); CA, CG, CK, CT, and CP, DNA amplified fromC.albicans(strain B311),C.glabrata(CDC Y-65),C.krusei(CDC 259-75),C.tropicalis(strain CDC 38),

andC.parapsilosis(ATCC 22019), respectively; B, water blank (negative control).

TABLE 2. Specificity of oligonucleotide probes to DNA from yeast-like fungid

Probea

Mean EI⫾SE (n) using DNA from:

H. capsulatum B. dermatitidis C. immitis P. brasiliensis P. marneffei S. schenckii C. neoformansb P. carinii Candida

speciesc

Dm 11.0⫾1.1 (34) 9.4⫾1.4 (24) 16.9⫾2.2 (14) 13.9⫾1.1 (21) 3.0⫾0.5 (31) 0 0 0 0

Hc 15.8⫾1.4 (37) 0 0 0 0 0 0 0 0

Bd 0 11.9⫾2.0 (20) 4.3⫾0.8 (10) 0 0 0 0 0 0

Ci 0 0 21.9⫾3.2 (13) 0 0 0 0 0 0

Pb 0 0 0 10.8⫾0.8 (21) 0 0 0 0 0

Pm 0 0 0 0 7.6⫾0.6 (36) 0 0 0 0

Ss 0 0 0 0 0 23.6⫾4.3 (12) 0 0 0

Cn 0 0 0 0 0 0 42.7⫾2.7 (31) 0 0

Pc 0 0 0 0 0 0 0 6.2⫾2.7 (10) 0

aSee Table 1 for definition of abbreviations.

bIncludes DNA from serotypes A, B, C, and D.

cCandidaspecies includedC. albicans,C. glabrata,C. krusei,C. tropicalis, andC. parapsilosis; excludes results for Hc probe againstC. albicans; mean EISE

6.5⫾1.0 (n⫽10).

dMean EIstandard error (n) for all heterologous DNA tested with the following probes: Dm 1.130.04 (n89); Hc, 1.20.3 (n118); Bd, 1.410.12 (n

103); Ci, 1.0⫾0.01 (n⫽96); Pb, 1.01⫾0.02 (n⫽88); Pm, 0.99⫾0.02 (n⫽98); Ss, 0.99⫾0.01 (n⫽103); Cn, 0.98⫾0.01 (n⫽95); and Pc, 0.98⫾0.01 (n⫽96).

All probes significantly hybridized to homologous DNA but not heterologous DNA atP⬍0.001 except for Pc (P⬍0.05) and Bd versusC. immitisDNA (P⬍0.01).

A value of 0 was assigned for all EI values less than 1.75 for ease of presentation.

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amplification and was then assessed by both agarose gel elec-trophoresis and PCR-EIA. Agarose gel elecelec-trophoresis and ethidium bromide staining allowed detection of amplicons at a concentration as low as 16 pg per reaction (Fig. 4; Table 4). In contrast, as little as 3.2 pg of DNA per reaction could be detected by PCR-EIA (Table 4).

DISCUSSION

This paper describes the development of a PCR-EIA that could amplify and identify DNA sequences from the rRNA gene of fungi that have yeast-like morphology in vivo. By use of universal fungal primers and a biotinylated universal probe, all fungal DNA was amplified and bound to strepavidin-coated microtiter plate wells. Identification of the fungi from which the DNA was isolated was then confirmed by microbe-specific oligonucleotide probes. The probes designed in this study could specifically identify DNA isolated from fungi that display yeast-like morphology in vivo. Our laboratory has had compa-rable success using a similar method for the identification of Candidaspecies (10, 11).

The sequential use of universal fungal primers for PCR amplification and microbe-specific probes to identify fungi has several advantages over methods used by others. The focus of most researchers has been to develop methods for the ampli-fication and identiampli-fication of a single species or genus of a

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TABLE 3. Reactivity of oligonucleotide probes to dimorphic pathogens against DNA from multiple strains of homologous and heterologous dimorphic fu ngi c Probe b Mean EI ⫾ SE ( n ) using DNA from a : H. capsulatum isolates B. dermatitidis isolates C. immitis isolates P. brasiliensis isolates 12 3 4 1 2 3 12123 Dm 13.7 ⫾ 2.8 (8) 8.8 ⫾ 1.8 (7) 10.3 ⫾ 2.0 (11) 11.4 ⫾ 2.8 (8) 9.2 ⫾ 1.8 (9) 12.5 ⫾ 3.3 (9) 4.9 ⫾ 0.8 (6) 14.8 ⫾ 1.8 (6) 18.5 ⫾ 3.6 (8) 13.3 ⫾ 2.2 (7) 14.1 ⫾ 2.3 (7) 14.3 ⫾ 2.0 (6) Hc 17.3 ⫾ 3.8 (9) 15.1 ⫾ 2.3 (6) 15.8 ⫾ 2.6 (10) 14.7 ⫾ 2.6 (9 ) 0 0 0 00000 Bd 0 0 0 0 9.7 ⫾ 2.4 (6) 15.4 ⫾ 3.5 (9) 8.3 ⫾ 3.3 (5) 4.7 ⫾ 1.2 (5) 3.8 ⫾ 1.2 (5) 0 0 0 Ci 0 0 0 0 0 0 0 23.8 ⫾ 4.2 (6) 20.2 ⫾ 4.9 (7) 0 0 0 Pb 0 0 0 0 0 0 0 0 0 9.4 ⫾ 1.5 (7) 12.3 ⫾ 1.4 (7) 12.0 ⫾ 0.4 (6) a Isolates used: H. capsulatum 1, G186B; 2, B293; 3, Down ’s; and 4, FLs-1; B. dermatitidis 1, 4478; 2, A2; and 3, KL-1; C. immitis 1, C634; and 2, C735; and P. brasiliensis 1, Pb18; 2, rh; and 3, soil. b See Table 1 for de finition of abbreviations. c Mean EI ⫾ standard error ( n ) for heterologous DNA tested with the following probes: Hc, 1.2 ⫾ 0.05 ( n ⫽ 42); Bd, 1.74 ⫾ 0.27 ( n ⫽ 44); Bd without Ci DNA, 0.99 ⫾ 0.02 ( n ⫽ 34); Ci, 1.0 ⫾ 0.02 ( n ⫽ 42); and Pb, 0.99 ⫾ 0.03 ( n ⫽ 33). All probes signi ficantly hybridized to homologous DNA but not to heterologous, dimorphic DNA from areas of endeminity at P ⬍ 0.001. A value of 0 was assigned for all EI values less than 1.75 for ease of presentation.

FIG. 4. Agarose gel of titratedH.capsulatumDNA (Down’s strain) amplified by PCR. Lane 1, molecular size markers in base pairs (Am-pliSize molecular ruler; Bio-Rad, Hercules, Calif.). The number of picograms of DNA per reaction for lanes 2 to 8 was 20,000, 10,000, 2,000, 400, 80, 16, and 3.2, respectively. Limit of sensitivity of agarose gel electrophoresis and ethidium bromide staining, 16 pg per reaction.

TABLE 4. Evaluation of the sensitivity of PCR-EIA

DNA concna EIb Reading in agarose gelc

10,000 53.3 ⫹

2,000 50.4 ⫹

400 38.2 ⫹

80 13.9 ⫹

16 5.3 ⫹

3.2 2.2 ⫺

0.64 1.5 ⫺

0.128 1.2 ⫺

0.0256 1.1 ⫺

aConcentration is given in picograms per reaction.

bEI usingH. capsulatum(Down’s strain) DNA.

c⫹, visually positive band in gel after ethidium bromide staining.

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particular fungal organism (3, 14, 21, 26, 29, 30, 32, 33, 37). This was accomplished in some cases through the use of single-copy gene targets, such as theERG11gene forCandidaspecies (26, 30) and theURA5gene forC.neoformans(37), or by use of microbe-specific primers for the amplification and identifi-cation of a single infecting pathogen (3, 29, 37). In the present study, universal fungal primers directed to the highly conserved ITS1 and ITS4 regions of ribosomal DNA allowed amplifica-tion of all fungal DNA rather than that from only a single organism. A complete array of different fungi could be identi-fied following a single PCR amplification and the application of specific probes. The rRNA gene was chosen as an amplifi-cation target, not only because it contains binding sites for universal fungal primers but because the chromosome on which this gene is located contains approximately 100 gene copies (23) that serve as a “preamplification” step to increase amplicon yield and test sensitivity. Therefore, the use of uni-versal primers and a multiple-copy gene target has greater utility and sensitivity for the identification of fungi in clinically diverse specimens.

The EIA format described in this study also has advantages over methods used by others for amplicon detection. Some investigators detected amplicons produced by microbe-specific primers after electrophoresis in agarose gels and ethidium bromide staining. The presence of a band was considered a positive result for those using specific primers (3, 14, 21, 29, 33). However, specific identification of fungi using amplicon size alone is not generally recommended (28) since different fungi may produce similarly sized amplicons, as was noted in the present study. Alternatively, the presence of a unique banding pattern after restriction enzyme digestion of the PCR product was used for species identification (26). Although the use of restriction enzymes is rapid and provides increased specificity compared to gel electrophoresis, results may be dif-ficult to reproduce and can be expensive. Often, two or more enzymes may be required for adequate specificity (26). In ad-dition, each enzyme may need different conditions for optimal restriction activity (24). Others developed specific probes to obtain a final identification of the organism using time-con-suming and labor-intensive Southern blot or slot blot methods (9, 30, 32, 35, 36, 37). The slot blot method uses membranes that are stripped and reprobed sequentially each time that a probe for a different organism is to be tested (17, 35, 36). In contrast, the EIA is very rapid (3 h) and simple to perform, and unlike the slot blot method, all probes can be tested simulta-neously.

A unique method using universal primers was developed by Turenne et al. (38) for determining the exact size of amplified DNA using an automated fluorescent capillary electrophoresis system. However, it was difficult to conclusively differentiate some fungi from others using this method because the size of the amplicons produced was similar if not identical for more than one fungus. In addition, the cost of the necessary equip-ment would make it difficult to use this assay in smaller labo-ratories.

The development of the probes described in our study should not only provide a means to identify fungi in culture but should also aid in the histologic identification of fungi in clin-ical specimens. Application of these probes to fungi in tissue sections will allow the differentiation of truly invasive

organ-isms from simple colonizers. Further testing of larger numbers of organisms from pure culture and application to clinical specimens are planned.

Whereas the organisms under study have unique character-istics that often allow histologic identification, atypical tissue forms may resemble other fungi (15, 19). Therefore, methods other than physical characteristics are needed to confirm iden-tification. Multiple techniques may be employed to identify fungi in tissue using molecular probes. First, fungal DNA can be extracted from the tissue and PCR-EIA can be performed using methods similar to those described in this paper. Second, the use of these probes in an in situ hybridization procedure would allow for the localization of fungal DNA directly in the tissue. Finally, the combination of these two procedures, where the target DNA is amplified and probes are hybrid-ized in situ, may be employed. None of these methods should be considered mutually exclusive. When fungi are found in large numbers in tissue, the faster, more versatile DNA extraction and PCR-EIA method may be most useful. In instances where there are very few fungal elements present in the tissue, in situ hybridization may be more useful because very little DNA would be present and might get “lost” when one tries to extract it from the tissue. Keeping the DNA local-ized and “bringing the probe to it” may prove more advanta-geous. However, the universal aspects of the PCR-EIA would be lost since a single probe would have to be selected and run individually on each slide.

The advantage of the PCR-EIA method is its ability to amplify and detect small quantities of DNA. However, some tissue may contain very low quantities of DNA, below the level of sensitivity. The sensitivity of a PCR-based assay can be enhanced by various modifications of the technique. First, one may use nested PCR (28, 33). This employs a second set of primers internal to the original set of primers to reamplify the target DNA using the amplicons from the first PCR as a tem-plate for the second PCR. This method has been shown to enhance test sensitivity (28, 33). Extreme care must be taken, however, so as not to contaminate reagents or the laboratory with amplicons from the first amplification. Second, one may continue the PCR through more cycles, continuing the geo-metric increase of DNA amplified. New forms ofTaq polymer-ase have been developed that have increpolymer-ased stability and accuracy throughout an increased number of PCR cycles. Pre-liminary results from our laboratory indicate that increasing the number of PCR cycles may increase the sensitivity of the PCR-EIA (data not shown).

In conclusion, oligonucleotide probes for yeast-like fungi have been developed and evaluated in a PCR-EIA format. These probes have been shown to be sensitive and specific and able to identify DNA obtained from fungi in pure culture. These characteristics, along with the rapid and convenient EIA detection format and its potential for automation, make it useful for applications in the clinical laboratory setting.

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Figure

TABLE 1. Sequences of oligonucleotide primers and probes
FIG. 2. Diagram of PCR-EIA procedure. PCR product is heat de-natured and incubated with both a biotinylated capture probe (ITS3-B)
FIG. 3. Agarose gel of amplified products from yeast-like fungi. Lane abbreviations (left to right): MW, molecular weight markers (Haeamplified fromamplified fromCK, CT, and CP, DNA amplified fromdigest ofandIII �X174 plasmid; Roche); H
TABLE 3. Reactivity of oligonucleotide probes to dimorphic pathogens against DNA from multiple strains of homologous and heterologous dimorphic fungic

References

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