JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 2011, p. 808–813 Vol. 49, No. 30095-1137/11/$12.00 doi:10.1128/JCM.01149-10Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Identification of Fusarium Species in Formalin-Fixed andParaffin-Embedded Sections by In Situ Hybridization

Using Peptide Nucleic Acid Probes�†Minoru Shinozaki,1 Yoichiro Okubo,1 Daisuke Sasai,1 Haruo Nakayama,1

Somay Yamagata Murayama,2 Tadashi Ide,1 Megumi Wakayama,1Nobuyuki Hiruta,1 and Kazutoshi Shibuya1*

Department of Surgical Pathology, Toho University School of Medicine, Tokyo, Japan,1 and Laboratory of Molecular Epidemiology forInfectious Agents, Graduate School of Infection Control Sciences, Kitasato University, Tokyo, Japan2

Received 7 June 2010/Returned for modification 21 July 2010/Accepted 17 November 2010

Fusarium has recently emerged as an opportunistic pathogen of humans, but the histological differentiationof Fusarium from Aspergillus and Scedosporium is particularly difficult because these fungi may induce similarclinical features and exhibit filamentous development in host tissues. Thus, there is a need to establish rapidand reliable methods that are applicable to pathological diagnoses. The aim of this study was to evaluate andestablish in situ hybridization (ISH) using peptide nucleic acid (PNA) probes targeting the 28S rRNA toidentify Fusarium species in tissue sections. This technique was validated using both formalin-fixed andparaffin-embedded pulmonary tissues from mice infected with seven different species of fungi and cell blocksfrom fungal cultures of 30 strains. As a result, strong positive signals were observed within fungal organismspresent in tissues of the lung from mice infected with Fusarium solani. Furthermore, this probe reacted stronglywith both F. solani and Fusarium oxysporum in sections from cell blocks. Although some cross-reactivityoccurred with the Pseudallescheria boydii in sections from cell blocks, the signal intensity was low and mosthyphae were not reactive. In conclusion, it was confirmed that ISH with PNA probes is accurate and is avaluable tool for identifying Fusarium spp. among organisms that have identical morphological features informalin-fixed and paraffin-embedded sections.

Early diagnosis of invasive fungal infection is essential be-cause the disease mostly occurs in patients with severely im-paired defense mechanism. Fusarium species are widely dis-tributed in soil, subterranean, aerial plant parts, plant debris,and other organic substrates, and mycotoxins produced bythese organisms have often been associated with animal andhuman diseases (16). In humans, Fusarium spp. cause a broadspectrum of infections, including superficial (keratitis andonychomycosis), locally invasive, and disseminated infectionsin immunocompromised patients (18). In addition to that in-vasive and disseminated infections caused by Fusarium spp. arebeing diagnosed with increasing frequency in patients withhematological malignancies (3), it has been accepted thatFusarium spp. are resistant to most antifungal agents (5).Therefore, an early diagnosis of the infection is now requiredto improve the outcome of treatment for seriously debilitatingconditions. Because of morphological similarities amongmolds in histopathological specimens, it has been difficult todifferentiate histologically Fusarium spp. from other molds.Recently, sensitive and rapid molecular detection assays thatuse PCR-based methods have been introduced to detect Fusar-ium DNA in serum, total blood, and tissue samples (10). How-

ever, there have been a few attempts to use in situ hybridization(ISH) to identify Fusarium spp. in tissue sections for histolog-ical diagnosis (8, 14). We are describing the first report of ISHusing peptide nucleic acid (PNA) as the probe targeting the28S rRNA of Fusarium spp. to identify the fungus in formalin-fixed and paraffin-embedded tissue sections that are widelyused as routine preparations for surgical and anatomical pa-thology in hospitals.

MATERIALS AND METHODS

Preparation of infected animals and tissue specimens. To verify the specificityof probes, sections of formalin-fixed and paraffin-embedded tissues of lung wereprepared from mice experimentally infected with seven different fungi. Lung hasbeen understood as one of the commonest organ involved by invasive fungalinfection. A part of this may be explained by the fact that the lung serves as aporta of infection. Therefore, in the present study, lungs from mice with intra-tracheal infection were used as a tissue specimen to evaluate the ISH procedure.

Six-week-old, male Institute of Cancer Research (ICR) mice (Sankyo LaboService Corp., Inc., Tokyo, Japan) were used in the present study. Immunesuppression was achieved by intraperitoneal injections of cyclophosphamide(Shionogi and Co., Ltd., Osaka, Japan) at a dose of 150 mg/kg (body weight) 3days prior to infection. To prevent bacterial infection, the animals were alsointraperitoneally administered with imipenem/cilastatin sodium (Banyu Pharma-ceutical, Tokyo, Japan). Prior to inoculation, the animals were anesthetizedintraperitoneally with 80 mg of ketamine (Daiichi Sankyo Co., Ltd., Tokyo,Japan) and 10 mg of xylazine (Bayer Health Care, Tokyo, Japan)/kg. Theconidiae or yeast cells were injected intratracheally as previously described (19).A 25-�l aliquot of the conidiae or yeast cell suspension was injected into thetrachea via a clinically used intravascular catheter (24G, Insyte-W; Becton Dick-inson, Hollister, CA). The mice were infected with 3 � 105 conidiae or yeast cellsof Aspergillus fumigatus (TIMM1776), Aspergillus terreus (TIMM2929), Aspergil-lus flavus (TIMM2935), Candida albicans, (TIMM1768), Rhizopus oryzae(TIMM1326), Fusarium solani (TIMM1303), and Pseudallescheria boydii(TIMM0952). The animals were sacrificed on the third day after infection, and

* Corresponding author. Mailing address: Department of SurgicalPathology, Toho University School of Medicine, 6-11-1 Omori-Nishi,Ota-Ku, Tokyo 143-8541, Japan. Phone: 81-3-3762-4151. Fax: 81-3-3767-1567. E-mail: kaz@med.toho-u.ac.jp.

† Supplemental material for this article may be found at http://jcm.asm.org/.

� Published ahead of print on 24 November 2010.

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the lungs were removed and fixed in 10% formalin, followed by dehydration withethanol and embedding in paraffin. Tissue sections (3 �m) were mounted onaminoalkylsilane-coated slide glasses (Dako Japan, Tokyo, Japan). Pulmonarylesions induced by this procedure were confirmed by histological examinationusing these sections stained with hematoxylin and eosin (H&E) and Grocott’sstains.

Strains tested. For specificity testing of the probes, cells from the following moldsand yeasts were tested: A. flavus var. flavus (NBRC 33021), Aspergillus niger (NBRC33023), A. terreus (NBRC 33026), A. fumigatus (NBRC 6344), A. fumigatus var.fumigatus (NBRC 33022), F. solani (NBRC 5232), Fusarium oxysporum (NBRC7152), P. boydii (NBRC 8078), R. oryzae (NBRC 5780), Cunninghamella elegans var.elegans (NBRC 4446), Rhizomucor pusillus (NBRC 9744), Mucor circinelloides f. sp.circinelloides (NBRC 4554), Penicillium commune (NBRC 5763), Pseudocochliobolusspicifer (NBRC 100222), C. albicans (ATCC 10231), Trichosporon asahii(CBS2479T), C. albicans var. stellatoidea (TIMM0310), Candida glabrata(TIMM1064), Candida guilliermondii (TIMM0260), Candida kefyr (TIMM0302),Candida krusei (TIMM0269), Candida lusitaniae (TIMM1668), Candida parapsilosis(TIMM0292), Candida tropicalis (TIMM0313), Cryptococcus neoformans(TIMM0354), Debaryomyces polymorphus (TIMM2937), Hansenula anomala(JCM3585), Pichia subpelliculosa (IFO0808), Saccharomyces cerevisiae(TIMM0925), and Schizosaccharomyces pombe (TIMM3376).

Preparation of cell blocks from cultured fungal cells. Mold strains were grownfor 48 to 72 h at 25°C in potato dextrose broth (Sigma Aldrich, St. Louis, MO).Yeast strains except Candida spp. were grown overnight at 25°C in YMPD broth(0.3% yeast extract, 0.3% malt extract, 0.5% peptone, 1.0% glucose) (BectonDickinson), and Candida spp. were grown overnight at 37°C in Medium 199(Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) with 10% calf fetal serum(Sigma-Aldrich Co., St. Louis, MO). From cultures of the above-mentionedmolds or yeasts, fungal cell suspensions were prepared in 10% formalin forfixation. Fixed molds were collected with centrifugation for 10 min at 2,000 rpm.

The cluster of molds was carefully transferred onto filter paper (Advantec Toyo,Ltd., Tokyo, Japan) with pointed forceps. This was also wrapped with same filterpaper and placed in an embedding cassette (Murazumi Industrial Co., Ltd.,Hyogo, Japan) and then penetrated with paraffin by using an automated tissueprocessor (Tissue-Tek VIP Premier; Sakura Finetek Japan Co., Ltd., Tokyo,Japan) (12). The cluster of molds penetrated with paraffin was transferred tobottom of an embedding stainless dish, followed by filling solidifying of paraffin,and cut into 3-�m sections that were then mounted on aminoalkylsilane-coatedslide glasses (Dako Japan, Tokyo, Japan).

To prevent the diffusion of yeast cells in suspension and to obtain a highdensity in paraffin blocks, we used agarose gel as an intermediate embeddingmedium. Yeast cells were collected with centrifugation for 10 min at 2,000 rpmand added with 50 �l of 2% liquid agarose at 65°C. This agarose gel was heapedonto the surface of cover glass (Matsunami Glass Ind., Ltd., Osaka, Japan), andsolidified at room temperature. The solidified gel was penetrated with paraffin byusing an automated tissue processor (Tissue-Tek VIP Premier), as well, andsections were prepared in same way (12).

PNA probes. The design of the PNA probes for Fusarium spp. (N terminus-GAT GAT CAA CCA AGC CCA) and panfungal species (N terminus-TACTTG TGC GCT ATC GGT) was derived from a comparison of 28S rRNA genesin the GenBank database. The alignment of the DNA sequence was performedby using Vector NTI Advance TM10 (Invitrogen, Carlsbad, CA). After align-ment and visual assessment of the Fusarium and non-Fusarium sequences, theprobe for Fusarium spp. was designed. As shown in Fig. 1, a Fusarium sp.antisense PNA probe targeting the 28S rRNA could be designed and evaluatedon a genus-specific level. The sequence revealed at least 9 to 14 mismatcheswithin the target region in sequences of nontarget organisms. Furthermore, toassess the retention of RNA in samples, we also designed a panfungal antisensePNA probe in the same way. Each selected sequence was checked for specificityagainst the GenBank database by using the Basic Local Alignment Search Tool

FIG. 1. Alignment of 28S rRNA sequences for Fusarium species, C. albicans, and other important hyalohyphomycetes for histologicaldifferentiation. The binding regions of the antisense probe used in the present study to detect Fusarium rRNA were boxed and magnified.Homologous regions were highlighted in gray. Species and GenBank accession numbers were as follows: C. albicans (AB436387), Absidia glauca(AF113447), Cunninghamella bertholletiae (AF113459), Mucor hiemalis (AF113468), Rhizopus oryzae (DQ466617), Ajellomyces capsulatus(AB176493), A. fumigatus (AB354577), A. terreus (AF454185), Penicillium expansum (AJ519347), P. boydii (EF151324), F. solani (AF178377), F.oxysporum (AF060383), and Fusarium verticillioides (U34526).

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(BLAST; http//www.ncbi.nlm.nih.gov/BLAST/). The selected sequence was thensynthesized, and the N terminus of the oligomer was conjugated to fluoresceinisothiocyanate (FITC) via a double aminoethoxyethoxyacetate (AEEA) linker(Fasmac Co., Ltd., Kanagawa, Japan).

ISH. The ISH procedure was performed as described previously (24). Briefly,sections were deparaffinized and rehydrated according to standard procedures.To expose target nucleic acids in the formalin-fixed tissue, the sections weretreated with a 1 mM concentration of EDTA buffer (pH 8.0) in a water bath(Thermo Fisher Scientific K.K., Yokohama, Kanagawa) for 20 min at 98°C anddigested with a 10-�g/ml concentration of proteinase K (Nippon Gene Co., Ltd.,Tokyo, Japan) for 10 min at 37°C. Hybridization was performed at 56°C for 90min with 1 �g of PNA probe/ml dissolved in hybridization medium (Dako Japan,Tokyo, Japan). After repeated washings with 2� standard saline citrate (SSC) at56°C, the signals were detected by enzyme immunohistochemistry using an anti-FITC antibody (Roche Diagnostics K.K., Tokyo, Japan) and horseradish perox-idase-labeled polymer solution (Nichirei Biosciences, Inc., Tokyo, Japan). Fi-nally, the sites of peroxidase were visualized by 3,3�-diaminobenzidinetetrahydrochloride (DAB; Dojindo Laboratories, Kumamoto, Japan) in the pres-ence of H2O2 and nickel and cobalt ions (1). As negative controls, ISH proce-dures were performed with a C. albicans PNA probe (20).

RESULTS

Paraffin sections of both lungs of mice infected with differentfungi and cell blocks mounted on the slide glasses were H&Eand/or Grocott’s stained, processed with ISH, and observedunder light microscopy for evaluation of our ISH procedure.

Specificity of ISH for Fusarium spp. in infected animal mod-els. Histological examination revealed an extensive fungalgrowth both in alveoli and terminal bronchus with necrosis andminor polymorphonuclear leukocyte infiltrate (Fig. 2). Theestablishment of experimental pulmonary lesions was con-firmed in mice infected with all seven of the different fungi thatwe examined.

Formalin-fixed and paraffin-embedded pulmonary tissuesfrom mice infected with seven different fungi were tested toassess whether the probe hybridized specifically with Fusariumspp. Strong positive signals against 28S rRNA of Fusarium spp.were observed within fungal organisms present in lung tissuefrom mice infected with F. solani (Fig. 3d). Positive organismstypically exhibited a signal visualized by a DAB reaction thatwas limited in a large part of cytoplasm and can be recognizedas black fine dots. The signal intensity varied within and be-tween fungal organisms in tissue sections. No substantial back-ground signal was observed in any tissue. In addition, no hy-bridization was found in other fungi tested.

Specificity of ISH for Fusarium spp. in cell blocks of cul-tured fungi. In total, 30 cell block sections from formalin-fixedand paraffin-embedded fungi of 30 strains were studied (seeFig. S1 in the supplemental material). Within the panel of 30fungi, the Fusarium sp. PNA probe reacted strongly with bothF. solani (Fig. 4d) and F. oxysporum in sections of cell blocks.The signal intensity and distribution in fungal organisms weresimilar to those observed in animal models. With the exceptionof the Fusarium spp., the P. boydii in sections of cell blocks (notof tissue sections) showed positive reactivity for the probe, buttheir signal intensity was low and most of the hyphae werenegative (Fig. 4c). No hybridization was observed in otherfungi tested.

Control experiments. To confirm the specificity of the 28SrRNA signals, adjacent sections were hybridized with a C.albicans PNA probe. A strong positive signal was detected in C.albicans in a tissue section, whereas no hybridization signal wasfound in the other fungi tested. The panfungal PNA probe

FIG. 2. Microphotographs of a pulmonary lesion in a mouse 3 days after intratracheal infection of F. solani (TIMM1303). (A) Histologicalexamination revealed an extensive fungal growth both in alveoli and terminal bronchus with necrosis and minor polymorphonuclear leukocyteinfiltrate with scattering nuclear debris (H&E stain; original magnification, �100). (B) There is extensive hyphal growth of invading mold showingdichotomous branching (Grocott’s stain; original magnification, �100).

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reacted with all fungi tested (Fig. 3e to h, Fig. 4e to h). Theintensity of ISH signals for the panfungal probe was similar tothat with species-specific probes.

DISCUSSION

Recently, several genome databases have provided new in-formation that can be used for field studies for the molecularidentification and epidemiology of pathogenic fungi. Accord-ingly, sensitive and rapid molecular detection assays have beenestablished by using PCR-based methods to detect fungalDNA (7, 11, 27). The application of these molecular tech-niques to formalin-fixed and paraffin-embedded tissue has alsobeen reported (2, 13, 21). Although there have been a fewattempts to use ISH to detect fungal agents in histopatholog-ical specimens (6, 8, 9, 14, 15), the use of ISH for the diagnosisof fungal infection in formalin-fixed and paraffin-embeddedsections has not been systematically assessed. We have previ-ously reported that a combination of high-temperature heatingin solutions of high pH, followed by a 10-min proteinase Kdigestion step, gave better ISH results (24). The heating pre-treatment used in the present study was adapted from antigenretrieval techniques used in conventional immunohistochem-istry (23).

Our purpose was to evaluate and establish an ISH procedurefor the detection of Fusarium spp. in formalin-fixed and par-affin-embedded sections. Diagnosis of fusariosis from culturesremains a difficult and time-consuming task, relying on mor-

phological and physiological examinations and requiring somedegree of expertise. Fusarium spp. are phylogenetically heter-ogeneous with variable antifungal susceptibilities (25). An ap-proach based on PCR methods has been used to detect Fusar-ium DNA (10). Although there have been a few attempts touse ISH to identify Fusarium spp. in histopathological speci-mens (8, 14), to our knowledge there has been no report ofISH using a PNA probe. Our results obtained with mice ex-perimentally infected with seven different fungi showed that F.solani can be specifically detected in infected tissues by ISHwith a PNA probe targeting 28S rRNA of Fusarium spp. Onthe other hand, using cell block sections from formalin-fixedand paraffin-embedded fungi, this probe reacted strongly withboth F. solani and F. oxysporum, but some cross-reactivity wasobserved in P. boydii hyphae. Part of this result may be ex-plained by the fact that P. boydii has a sequence similar to thetarget of our probe.

PNA molecules are DNA mimics in which the negativelycharged sugar-phosphate backbone is replaced by a neutralpolyamide backbone, formed by repetitive units of N-glycine.This structure enables PNA probes to hybridize to complemen-tary nucleic acid targets with high specificity and rapid bindingkinetics (4, 17). Due to the novel properties of its hybridiza-tion, PNA is beginning to be applied in ISH to detect fungalnucleic acids (20, 22, 26, 28); however, there has been noreport of the application of such a probe to formalin-fixed andparaffin-embedded tissues. Better outcomes are obtained with

FIG. 3. Specificity verification of the Fusarium sp. PNA probe and assessments of rRNA retention and its hybridizability in experimentallyinfected mice. The tissue sections were hybridized with Fusarium sp. PNA probe (a to d) or with panfungal PNA probe (e to h). Strong positivesignals against 28S rRNA of Fusarium spp. were observed in lung tissues from mice infected with F. solani (d). The panfungal PNA probe reactedwith all fungi tested (e to h). (a and e) A. fumigatus (TIMM1776); (b and f) A. terreus (TIMM2929); (c and g) P. boydii (TIMM0952); (d and h)F. solani (TIMM1303). Original magnification, �400.

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PNA probes compared to conventional DNA probes (26). Inour first approach, we confirmed that PNA probes requiredshorter hybridization times than double-stranded DNAprobes. From the standpoint of decreased assay turnaroundtime, the application of PNA probes is especially attractive.

Recently, Montone reported that the use of dual fluoro-genic-labeled locked nucleic acids (LNA) probes of ISH wereable to differentiate Fusarium from Aspergillus organisms (14)and that the LNA probe produced a stronger signal comparedto a DNA probe with the same sequence (15). Our probe coulddifferentiate Fusarium from 23 fungal species other than As-pergillus. These novel findings demonstrate the feasibility ofthe approach and strongly suggest that LNA and PNA can bewidely used as probes of ISH in the near future.

The 28S rRNA sequence was selected as a detection targetbecause its large size may reveal adequate differences in dis-tinguishing closely related organisms. In addition, it has beenaccepted that multiple copies of ribosomal genes are present infungi, which can be transcribed into rRNA. It is essential thatassessment of retention of rRNA and its hybridizability shouldbe performed, because loss of rRNA or failure of the accessi-bility of probes in processed tissue sections can lead to mis-leading results. In the present study, we designed a panfungalPNA probe and confirmed that the intensity of ISH signals ofthis probe was similar to those of species-specific probes.These findings suggest that ISH with the panfungal probe may

be useful for the estimation of hybridizable rRNA for thespecific detection of human pathogenic fungi.

In conclusion, we have shown the superiority and the use-fulness of ISH with PNA probes for identifying Fusarium spp.in formalin-fixed and paraffin-embedded sections. Furtherstudies are needed to establish ISH with PNA probes as anaccurate and rapid diagnostic procedure for tissue sectionsfrom patients with suspected fusariosis.

ACKNOWLEDGMENTS

This study was supported by Health Science Research grants forResearch on Emerging and Re-Emerging Infectious Diseases (H16-Shinko-6, H19-Shinko-8, and H22-Shinko-8) and Measures for Intrac-table Diseases (H20 Nannchi Ippann 35) from the Ministry of Health,Labor, and Welfare of Japan and by the Grant of the Strategic Basis onResearch Grounds for Non-Governmental Schools at Heisei 20th fromthe Ministry of Education, Culture, Sports, Science, and Technology ofJapan to K.S.

We are grateful to K Makimura, K Uchida, and H Yamaguchi forkindly providing important advice.

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