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00019606-201006000-00006ArticleDiagnostic Molecular PathologyDiagnostic Molecular Pathology© 2010 Lippincott Williams & Wilkins, Inc.19June 2010 p 99-104In Situ Hybridization for Coccidioides immitis 5.8S Ribosomal RNA Sequences in Formalin-fixed, Paraffin-embedded Pulmonary Specimens Using a Locked Nucleic Acid ProbeA Rapid Means for Identification in Tissue SectionsOriginal ArticlesMontone, Kathleen T. MD*; Litzky, Leslie A. MD*; Feldman, Michael D. MD,PhD*; Peterman, Heather MD*; Mathis, Benjamin MD*; Baliff, Jeffrey MD*; Kaiser, Larry R. MD†; Kucharczuk, John MD†; Nachamkin, Irving Dr PH, MPH**Department of Pathology and Laboratory Medicine†Department of Surgery, Division of Thoracic Surgery, University of Pennsylvania, Philadelphia, PAReprints: Kathleen T. Montone, MD, Department of Pathology and Laboratory Medicine, Hospital of the University of Pennsylvania, 3400 Spruce Street, 6 Founders, Philadelphia, PA 19104 (e-mail: [email protected]).AbstractCoccidioides immitis/Coccidioides posadasii are common causes of pulmonary infection in certain geographic areas, and are highly infectious when working with culture isolates in the laboratory. Rapid techniques to accurately identify this pathogen in tissues may be of benefit for diagnosis and in limiting the exposure of laboratory personnel to this agent. Locked nucleic acids (LNA) are modified nucleotides in which a ribonucleoside is linked between the 2′-oxygen and the 4′-carbon atoms with a methylene unit. LNA oligonucleotides exhibit increased thermal stability and make excellent probes for in situ hybridization (ISH). In this study, ISH utilizing a biotin-labeled LNA probe targeting Coccidioides sp. ribosomal RNA sequences in 6 formalin-fixed, paraffin-embedded pulmonary tissue specimens from 6 patients with culture positive or histologic findings suggestive of Coccidioides sp. infection is described. The cultures of the pulmonary specimens confirmed C. immitis in 3 of 6 patients. The ISH procedure with the LNA probe was positive in all 6 cases, although the number of organisms that were highlighted varied from rare to numerous. ISH with a biotin-labeled DNA probe of the same sequence was positive in 4 of the 6 cases and the signal intensity and number of organisms was much less than that observed with the LNA probe. Negative control tissues containing a variety of different fungal pathogens including Aspergillus sp., Fusarium sp., Blastomyces dermatitidis, Candida sp, Histoplasma capsulatum, and Zygomyces did not hybridize with the LNA and DNA probes. ISH with an LNA oligonucleotide probe targeting Coccidioides sp. ribosomal RNA is useful for rapid ISH. ISH could be rapidly performed when fungal pathogens are observed in tissue but cultures are negative or have not been performed.Coccidioides immitis and Coccidioides posadasii are highly infectious fungal pathogens most commonly causing pulmonary infection, but they may also cause disseminated infections in some patients. The organisms are most commonly found in soil in warm, dry areas, particularly in Mexico, the southwestern US and Southern California; infection is rare in the northeastern US.1 After inhalation of spores, exposed patients often have a mild respiratory illness and acquire immunity from the disease, although in some individuals disease can be extremely severe. After exposure, the most common clinical presentation of disease is as a solitary pulmonary nodule that is usually identified incidentally in a patient undergoing cancer screening. Owing to its virulence and production of small arthrospores that are highly infectious, Coccidiodes sp. is considered to be a danger when grown in the clinical microbiology laboratory.2 Rapid techniques to accurately identify this pathogen in tissues may be of benefit for diagnosis and in limiting exposure of laboratory personnel to this highly infectious agent.Ribosomal RNA (rRNA) are conserved, abundant species-specific sequences that are used for phylogenetically classifying organisms including fungi such as Coccidioides sp.3,4 Owing to their abundance and species specificity, rRNA sequences have been established as optimal targets for hybridization reactions including in situ hybridization (ISH).5Locked nucleic acids (LNA) are modified nucleotides in which the 2′ oxygen and the 4′ carbon are linked through a methylene unit.6–8 This change results in a “lock” on the nucleotide.6–8 LNA nucleotides can be incorporated into RNA or DNA nucleic acid probes. These special nucleotides hybridize strongly to their complementary RNA and DNA nucleotides, producing hybrids that are thermally stable.6–8 Limited studies have used LNA probes to detect bacterial rRNA sequences by fluorescent ISH in single-cell preparations, and more recently our laboratory reported an ISH assay for Aspergillus sp. using LNA probes targeting 18S rRNA sequences.9–11 The utilization of LNA nucleic acid probes in detecting rRNA of Coccidioides sp. organisms has not been reported earlier. In this study, a test using a biotin-labeled LNA probe targeting Coccidioides sp. 5.8S rRNA sequences in formalin-fixed, paraffin-embedded tissues is described.MATERIALS AND METHODSPatient MaterialSix formalin-fixed, paraffin-embedded pulmonary specimens from 6 patients with histopathologic evidence of fungal infection strongly suggestive of Coccidioides sp. were obtained from the surgical pathology files at the Hospital of the University of Pennsylvania. The hematoxylin and eosin slides and Grocott silver stains were reviewed. Four-micron sections were cut from the paraffin blocks and placed on ProbeOn Plus slides (Fisher Scientific) for use in the ISH procedure. This study was approved by the University of Pennsylvania Institutional Review Board.Nucleic Acid Probes (Table 1)LNA Probes (Table 1)A synthetic 20-mer with the sequence 5′-CTCTTTTTTTTATTATATCC-3′ was commercially synthesized using 55% DNA nucleotides and 45% LNA nucleotides, and was high-performance liquid chromatography (HPLC)-purified and 3′ terminally biotin-labeled with a single biotin-tetra-ethyleneglycol (TEG) 15-atom spacer arm (Sigma Genosys). This probe sequence has been previously reported to detect C. immitis 5.8S rRNA sequences through a PCR-enzyme immunoassay procedure12 and has 100% homology to C. immitis and C. posadasii but not to other fungal rRNA sequences by BLAST analysis (GenBank). The lyophilized probe was diluted to 1 ug/uL using Tris-ethylenediaminetetraacetic acid (EDTA) buffer, pH 8.0 (Amresco, Solon, OH). On the basis of previously established studies,13,14 the probe was diluted to a final concentration of 1 ug/mL in a nonformamide-based probe diluent (675 mM NaCl, 138 mM sodium citrate, 12 mM sodium phosphate dibasic, 63 mM sodium phosphate dibasic, 34 mM ascorbic acid, 15 mM sodium azide, 5 mM EDTA, and 0.5% chondroitin sulfate) for the ISH procedure15 and used for the ISH studies.JOURNAL/dimp/04.03/00019606-201006000-00006/table1-6/v/2021-02-17T200020Z/r/image-tiffCoccidioidies sp. rRNA Nucleic Acid Probes SequencesTo determine whether the fungal rRNA was conserved in the tissue specimens, a 20-mer probe with the sequence 5′CCGATCCCTAGTCGGCATAG-3′ was synthesized using 55% DNA and 45% LNA nucleotide, was HPLC-purified and 3′ biotinylated in a fashion similar to the Cocciodioides sp. probe (Sigma Genosys). This LNA probe has been previously used to detect pan-fungal rRNA sequences in tissue sections by ISH.11,14–16 In addition, as a negative control, ISH with an LNA probe targeting Aspergillus 18S rRNA sequences was performed on the confirmed/suspected C. immitis tissues as reported earlier.11DNA Probes (Table 1)A DNA oligonucleotide probe of the same nucleic acid sequences as the LNA C. immitis/C. posadasii probe was synthesized, HPLC-purified and 3′ biotin-labeled with biotin-TEG (Integrated DNA Technologies). The lyophilized probe was diluted to 1 ug/uL using Tris-EDTA buffer, pH 8.0 (Amresco). This probe was also used at the same final concentration as the LNA probe for ISH (1 μg/mL). Two additional DNA probes targeting C. immitis 18S and 28S rRNA sequences were commercially synthesized and 3′ biotin labeled with biotin-TEG. These 2 sequences have been reported earlier to detect C. immitis sp. rRNA sequence by ISH in tissue sections using digoxigenin-labeled DNA probes,16 and were used together for ISH at a concentration of 1 μg/mL each.ISH ProcedureISH with the LNA probes was performed using modifications of previously described methods using manual capillary action technology on the MicroProbe Staining System (Fisher Scientific).17,18 In brief, the tissue sections were dewaxed, cleared, and rehydrated. The tissues were then digested in pepsin solution used at full strength (Diagnostic Biosystems) for 3 minutes at 105°C. As reported earlier, the short period of using pepsin at a high temperature does not seem to denature the effects of the enzyme, and results in uniform ISH results.19 The slides were washed with distilled water, the probe was applied to the slides and the tissues were heated at 105°C for 4 minutes to denature any secondary rRNA structures, cooled at room temperature for 1 minute and then hybridized at 50°C for 1 hour. After hybridization, the tissues were washed with 2X standard saline citrate 3 times at 2 minutes each at 50°C. The hybrids were detected with prediluted streptavidin conjugated to alkaline phosphatase (Biogenex) for 30 minutes at room temperature followed by signal development with nitroblue tetrazolium chromogen (Roche) for 30 minutes at room temperature. The slides were washed with distilled water, coverslipped, and examined by light microscopy. The method for the DNA probes was similar, with the exception that hybridization was carried out at 37°C and posthybridization washing was carried out using 2X standard saline citrate at room temperature.Negative ControlsNegative controls consisted of formalin-fixed, paraffin-embedded specimens containing Aspergillus sp., Fusarium sp., Blastomyces dermatitidis, Candida sp., Histoplasma capsulatum, and Zygomyces.Slide ExaminationAll slides were examined by light microscopy. Results were analyzed with regard to intensity using a scale of 1+ (weak), 2+ (moderate), and 3+ (strong) and the percentage of visible organisms staining (1+<30%, 2+30% to 60%, and 3+>60%).RESULTSThe clinical information on the 6 patients is presented in Table 2. All 6 pulmonary nodules were removed using video-assisted thoracoscopic surgery. Of the 6 patients, 4 had a prior diagnosis of a malignancy and the nodules were identified on routine imaging for follow-up to rule out metastatic disease. The remaining 2 patients had no known malignancy but presented with cough. Patient 4 also presented with hemoptysis, suggesting an involvement of the airway. Cultures of all 6 nodules were taken in the operating room.JOURNAL/dimp/04.03/00019606-201006000-00006/table2-6/v/2021-02-17T200020Z/r/image-tiff Results of ISH for Coccidioides immitis/Coccidioides posadasii in Lung NodulesOn histologic sections, all 6 specimens had granulomatous inflammation with extensive necrosis and evidence of fungal organisms on Grocott silver stains histologically suggestive of Coccidioides sp. The organisms were found in the spherule form with intact and ruptured cysts containing the fungal endospores. In addition, several cases contained arthroconidial forms. In 1 patient, there was a 2.0-cm cavitary mass with a necrotizing broncentric inflammatory reaction containing hyphal forms of the fungus adjacent to the spherules and arthoconidia. Cultures from the nodule grew C. immitis in 3 of the 6 patients, whereas the remaining 3 patients had negative cultures.ISH with the pan-fungal rRNA probe confirmed the preservation of fungal rRNA in all patients, but the number of organisms varied from rare strong positive to numerous strong positive organisms. The results of the ISH are presented in Table 2. The LNA Coccidioides sp. probe was positive in all 6 cases (Fig. 1). Not all histologically apparent organisms were detected with the assay in all cases (Table 2). Compared with the LNA probe, the DNA probe of the same sequence produced a weaker signal with fewer organisms staining in only 4 of 6 cases (Fig. 1). Two cases were negative with the 5.8S DNA probe. Using the cocktail 18S/28S DNA probes, fungal organisms were observed in all 6 cases (Fig. 1). In some cases staining was strong and was comparable to the LNA probe, but in others the signal produced was overall weaker (Fig. 1).JOURNAL/dimp/04.03/00019606-201006000-00006/figure1-6/v/2021-02-17T200020Z/r/image-jpeg In situ hybridization for Coccidioides sp. 5.8S ribosomal RNA (rRNA) with biotin-locked nucleic acids probe (A, D, G) in comparison with a biotin-labeled DNA probe of the same sequences (B, E, H) and a mixture of biotin-labeled DNA probes targeting 18/28S rRNA sequences (C, F, I). Original magnification ×40, nitro blue tetrazolium.ISH with all probes was negative in the control specimens that consisted of tissues containing a variety of different fungal pathogens such as Aspergillus sp., Fusarium sp., B. dermatitidis, Candida sp, H. capsulatum, and Zygomyces (Fig. 2). In addition, ISH with the Aspergillus 18S rRNA LNA probe was negative in all cases.JOURNAL/dimp/04.03/00019606-201006000-00006/figure2-6/v/2021-02-17T200020Z/r/image-jpeg In situ hybridization with the locked nucleic acids Coccidioides sp. probe in formalin-fixed, paraffin-emedded control tissues of Aspergillus fumigatus (A) and Blastomyces dermatitidis (B). Original magnification ×40, nitro blue tetrazolium.DISCUSSIONFungal rRNA sequences are useful molecular targets for species identification because within the highly conserved rRNA genes, there are regions of species-specific sequence signatures. ISH with DNA oligonucleotide probes is a means for providing tissue-based detection of a variety of fungal pathogens in formalin-fixed, paraffin-embedded tissues.11,14–18,20–22 Although culture is often considered the gold standard for fungal identification, ISH is helpful when cultures are negative or have not been performed and a fungal pathogen is histologically observed.LNA probes are excellent for ISH procedures.23,24 LNA-DNA and LNA-RNA hybrids are much stronger than either RNA-DNA or RNA-RNA hybrids.6–8 The hybrids produced with LNA probes are thermally stable and can withstand stringent conditions such as high temperatures and low-salt conditions.6–8 As a result, probes of short length can be effectively used. The number of studies that have used LNA probes in detecting rRNA sequences of infectious agents has been limited.9,10 We recently showed that an LNA probe specific to Aspergillus 18S rRNA sequences resulted in a signal twice as strong as a DNA probe of the same sequence.11 This study set out to compare ISH for C. immitis/C. posadasii rRNA sequences with LNA and DNA probes.ISH proved to be an excellent means for confirming the presence of Coccidioides sp. organisms in tissue sections. In fact, in this study all of the LNA and DNA probes used were able to confirm the presence of Coccidioides sp. organisms at least focally in the majority of histologically suspected cases of Coccidiomycosis, whereas culture only confirmed C. immitis in 50% of the cases. The biotinylated LNA probe targeting the 5.8S rRNA of C. immitis/C. posadasii had a clear advantage over the biotinylated DNA probe of the same sequence using the ISH procedure. Staining was consistently stronger and highlighted more organisms. The superior advantage of the LNA probe was less apparent when compared with the cocktail of biotin-labeled DNA probes designed to target the 18S and 28S rRNA sequences of C. immitis. In fact, in some specimens the staining intensities and the number of organisms highlighted were similar. It is possible that increasing the hybridization time or probe concentration or even the utilization of signal enhancement techniques such as catalyzed reporter deposition (CARD) (tyramide signal amplification techniques) or silver intensifications techniques could greatly enhance the ISH signal with the DNA probes. In fact, Hayden et al16 reported a sensitivity of 94.3% and a specificity of 100% using the 18S/28S C. immitis DNA probes in combination with either 3-hour hybridization or overnight hybridization with the CARD technique for cases that were initially negative by routine ISH. Using a similar approach including overnight hybridization and the CARD technique, Abbott et al25 were able to detect a variety of fungal pathogens including C. immitis in cutaneous specimens. In our experience, increasing the probe concentration 4-fold and increasing the hybridization time to 4 hours did not enhance the signal obtained with the LNA or the DNA probe used in this study (data not shown).An interesting feature of the study was that not all of the histologically evident fungal forms were highlighted by the ISH assay in some of the cases, regardless of the probe used. Even the LNA pan-fungal probe did not highlight all of the pathogens in the tissue sections, indicating either a lack of rRNA preservation or inaccessibility of the probe to the rRNA targets in the fungal organisms. As not all organisms may be detected by ISH, this method may have diagnostic limitations, particularly when used in needle biopsy or cytologic preparations that may only contain a few organisms, and therefore testing could result in a false negative. The reason why all of the C. immitis organisms were not highlighted is unknown. The use of microwave pretreatment with citrate buffer, higher probe concentrations (4 μg/mL), different proteinase pretreatments with higher pepsin concentrations or even alternative proteases (ie, proteinase K), longer hybridization times (4 hours) or silver intensification techniques (EnzMet; Nanoprobes, Inc.) did not seem to increase the number of C. immitis organisms in the tissue sections (data not shown). There are a few potential explanations for this reduced staining. It is possible that there is a spatial arrangement of the fungal rRNA within the organisms and therefore that the rRNA may not always be accessible in the tissue sections.26 It is also possible that the ISH assay detects only viable organisms, and that many of the C. immitis organisms in the tissue are not viable. In previous study, our group has shown that ISH for fungal pathogens is often weaker in entangled masses of fungi (fungus ball) and often shows more intense staining in the periphery of these fungus balls and on the leading edge of tissue invasion where the fungal organisms are more likely viable.20 In addition, Hayden et al16 observed reduced sensitivity for H. capsulatum detection by ISH in tissues with extensive necrosis in comparison with tissues without extensive necrosis. Many of the cases in our study showed extensive necrosis with scattered fungal organisms. It is also distinctly possible that the LNA and DNA probes are just not capable of penetrating the C. immitis cell walls with the methods used in this study. In foam samples, Carr et al27 observed an enhanced rRNA fluorescent ISH signal for bacteria containing mycolic acid in their cell walls using a variety of different pretreatments conditions. Despite what this group found to be their most optimal pretreatment (acid hydrolysis with lipase and proteinase K), they still encountered organisms without a signal, and hypothesized that absence of ISH signal may not always be explained by lack of probe penetration, but could be related to organism viability.As histopathology provides a relatively reliable means for diagnosing Coccidiomycosis (ie, all suspected cases of Coccidioides sp. were effectively confirmed by ISH), one may argue the necessity of a technique such as ISH for species identification. However, the diagnosis of Coccidiomycosis is not always straightforward. C. immitis/C. podasasii is a dimorphic fungus that has 2 forms: a hyphal form and a spherule form. The hyphal forms are rare in human tissues because they are most commonly observed at room temperature (25°C), although they may be present under other conditions, whereas the spherules are observed in the tissue phase and only produced in culture under special growth conditions. One of the culture-confirmed cases in our series had both spherule and hyphal forms. This patient had a large cavitary lesion and within the cavity were hyphal forms of the fungus that clearly invaded into tissue. If spherules were not evident in this case, a diagnosis of Coccidiomycosis would be difficult without ancillary techniques. Coccidiomycosis is often difficult to distinguish from other pathogens, particularly B. dermatitidis in early growth phases and the hyphomycetes, especially if arthroconidia are present and spherules are not yet evident.28 As the cultures in our series were positive only 50% of the time, the ISH provided a way in which accurate species identification could be achieved. It is quite important to confirm Coccidiomycosis. This pathogen is considered to be one of the most virulent and highly contagious fungal pathogens, particularly in immunosuppressed patients. It should only be grown in culture under biocontainment conditions, as the exposure of laboratory personnel to arthrospores has resulted in laboratory-acquired infections.2In summary, using an LNA probe targeting C. immitis/C. posadassi 5.8S rRNA sequences, we have developed a rapid ISH procedure for C. immitis/C. posadassi. When compared with DNA probes, the LNA probe reliably produced a stronger signal. 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Original magnification ×40, nitro blue tetrazolium. In situ hybridization with the locked nucleic acids Coccidioides sp. probe in formalin-fixed, paraffin-emedded control tissues of Aspergillus fumigatus (A) and Blastomyces dermatitidis (B). Original magnification ×40, nitro blue tetrazolium.In Situ Hybridization for <em xmlns:mrws="http://webservices.ovid.com/mrws/1.0">Coccidioides immitis</em> 5.8S Ribosomal RNA Sequences in Formalin-fixed, Paraffin-embedded Pulmonary Specimens Using a Locked Nucleic Acid Probe: A Rapid Means for Identification in Tissue SectionsMontone Kathleen T. MD; Litzky, Leslie A. MD; Feldman, Michael D. MD,PhD; Peterman, Heather MD; Mathis, Benjamin MD; Baliff, Jeffrey MD; Kaiser, Larry R. MD; Kucharczuk, John MD; Nachamkin, Irving Dr PH, MPHOriginal ArticlesOriginal Articles219p 99-104