Advances in Anatomic Pathology:
In Situ Hybridization for rRNA Sequences in Anatomic Pathology Specimens, Applications for Fungal Pathogen Detection: A Review
Montone, Kathleen T. MD*; Guarner, Jeannette MD†
*Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA
†Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA
All figures can be viewed online in color at http://http://www.anatomicpathology.com.
The authors have no funding or conflicts of interest to disclose.
Reprints: Kathleen T. Montone, MD, Department of Pathology and Laboratory Medicine, University of Pennsylvania, 3400 Spruce Street, 6 Founders, Philadelphia, PA 19104 (e-mail: firstname.lastname@example.org).
Fungal infections are a frequent occurrence in medical practice due to increasing numbers of immunosuppressed patients. New antifungal medications have been developed and it has become evident that different fungi require different treatments as some are intrinsically resistant to these drugs. Thus, it is imperative that pathologists recognize the limitations of histopathologic diagnosis regarding speciation of fungal infections and advocate for the use of different techniques that can help define the genus and species of the fungus present in the specimen they are studying. In this review we present the use of in situ hybridization as an important adjunct for the diagnosis of fungal diseases, the different techniques that have been used for fungal identification, and the limitations that these techniques have.
All prokaryotes (bacteria) and eukaryotic cells contain ribosomal RNA (rRNA) sequences which are highly conserved. rRNA is the central part of ribosomes and essential for protein synthesis. Each ribosome has 2 subunits, a large and a small and the size depends on the organism having a defined nucleus. The large rRNA in prokaryotes is 50S and contains a 5S and a 23S rRNA and in eukaryotes it is 60S containing 5S, 5.8S and 28S rRNA. The small rRNA in prokaryotes is 30S and contains 16S rRNA and in eukaryotes it is 40S containing 18S rRNA.1,2 rRNA sequences are the most conserved of nucleic acid sequences and have proven useful for phylogenetic classification of animal species.3 For example, through rRNA analysis Pneumocystis jirovecii (formerly Pneumocystis carinii) has been reclassified as a fungus.4 Analysis of rRNA has also been widely used in microbiology for identifying an infectious agent in entities thought to be infectious but in which no agent had been cultured. For example, through 16S rRNA analysis Bartonella henselae was found to be the agent causing bacillary angiomatosis and Tropheryma whippelli the agent responsible for Whipple disease5,6
Although portions of rRNA sequences are very conserved, there are portions that are species specific allowing the field of medical microbiology to grow as rRNA analysis has been explored. In 1985, Lane et al7 reported that sequencing 5S rRNA could be a useful tool for phylogenetically classifying bacterial organisms. Olsen and colleagues expanded that concept indicating that 16S and 23S rRNA sequencing was even more robust for organism classification because the longer sequences could allow for more specific organism classification.8,9 They also proposed that rRNA use in in situ hybridization (ISH) assays could be a useful tool for counting and identifying organisms in ecologic samples. Using ISH techniques, separate reports by Giovanni et al10 and Delong et al11 described general and specific approaches to detection of bacteria including identification of multiple bacteria on a sample placed in a single slide. Although these methodologies were first applied in the field of environmental microbiology and ecology, their eventual adaptation into medical microbiology was inevitable.
rRNA sequences are prime targets for hybridization reactions for infectious agents for several reasons.12 First of all the structure of rRNA, although complex, is similar to other nucleic acid sequences having a 5′ and a 3′ end and sequences are largely known or discoverable, therefore probes can be easily produced. In addition, rRNA sequences are widely conserved even though regions are species specific. rRNA sequences are usually abundant, especially in Eukaryotic cells where rRNA makes up at least 80% of RNA molecules and therefore are excellent for hybridization reactions.1,2
The application of rRNA ISH has been quite numerous including applications to environmental samples such as bacterial and fungal detection in soil and water, identification of known, unknown, and unculturable bacterial specimens, and more recently specific determination of microorganisms in tissue and cytologic specimens, blood, and biofilms. Although these techniques have been described and can be used in the diagnostic setting, they have not been adopted widely in anatomic pathology departments as they are considered laboratory-developed molecular assays and need extensive validation13
WHY UTILIZE IN SITU HYBRIDIZATION FOR ORGANISM rRNA SEQUENCE DETECTION
There are multiple instances in which performing ISH to detect rRNA of organisms in pathologic specimens is of diagnostic importance. First of all the pathologist may see a microorganism in the specimen but a culture is not available because of growth requirements that were not met for the organism, no organisms are present in the specimen sent for culture, or all the specimen was preserved in such a way that culture is not possible (all tissue in formalin). Second, the specimen may have been very small and a decision to use it all in the test that would provide the highest yield for the amount of material was taken, placing the entire specimen in formalin for pathologic study. There may be a discrepancy between what the culture is growing and the organism present in the pathologic section. Finally, the cultures may grow an organism that cannot be found in the pathologic specimen or the pathology observed is not what one expects for the organism cultured. The earliest targets for rRNA ISH included assays for bacteria, fungi, and parasites with some assays being performed in under 60 minutes.14–18 While a variety of ISH assays for rRNA targets have been developed, the remainder of this review will describe ISH for fungal rRNA sequences.
WHY DEVELOP IN SITU MEANS FOR FUNGAL DETECTION?
Several years ago, there were very few agents that could be used for effectively treating invasive fungal infections. As such, the genus and species of the fungus cultured was often clinically insignificant since very few antifungal agents were available. More recently, many new antifungal agents have been developed, the toxicity of these agents has decreased, and more importantly, some fungal genus and species are resistant to some of the antifungal agents.19 Because of this, it has become extremely important to determine the genus and if possible species of fungus involved in a disease process, in order to guide patient management.
In tissue sections, fungal infections can be divided into 2 groups, the yeasts and the molds.20 The molds or filamentous fungal organisms can be further divided into 3 groups, those that have melanin which include the pigmented or dematiaceous fungi, those that show frequent septations which include Aspergillus, Fusarium and many other species, and those that have rare septations or pauciseptated fungi which include the Mucor genera or zygomycetes. Unfortunately, molds are often misdiagnosed as pigment may not be prominent or few septations may be overcalled histologically. Sangoi et al21 observed histologic misidentification of fungal forms in 21% of patients diagnosed with systemic fungal diseases (with a potential adverse clinical outcome in 2 of 8 patients with available follow-up). Of 10 patients in which organisms were misclassified on histologic examination, 4 were interpreted as Aspergillus by histopathology. By culture, these 4 cases grew Rhizopus (1 case), Fusarium (1 case), and Scedosporium apiospermium (2 cases). Similarly, Lee et al22 observed discordance between histology and culture results in 17% of patients with the greatest difficulty seen in the identification of nonpigmented (hyalohyphal) fungi which are most commonly diagnosed as “consistent with Aspergillus” (Fig. 1). However, it should be noted that neither of these publications considered the possibility of dual fungal infections.
The yeasts include the thermally dimorphic fungi (Blastomyces dermatitidis, Coccidioides immitis, Paracoccidioides brasiliensis, Sporothrix schenckii, Histoplasma capsulatum, and Penicillium marneffei) and those fungi which only exist as yeast such as Cryptococcus and Candida glabrata. Although there are certain morphologic characteristics such as size, budding pattern (broad-based vs. narrow-based, and number of buds), presence of a mucous capsule, and grouping (spherule with endospores or clustering) that can help in guiding the possible fungal species, misdiagnosis occur. Patel et al23 performed a retrospective study of 53 patients with pathologic evidence of broad-based budding yeasts which is characteristic of Blastomyces; however, C. immitis, Candida albicans or Aspergillus were recovered from 4 (10%) of these specimens. Lemos et al24 also showed that culture overgrowth with Candida was frequent in patients with blastomycosis.
Probably the fungi that are most frequently misdiagnosed histopathologically are C. albicans and other Candida spp. as in tissues they can produce yeasts and a filamentous component (pseudohyphae and hyphae). When Candida spp. are found on the mucosal surface of the gastrointestinal tract and female genital tract, there is usually no confusion. However, invasive candidiasis may appear as hyaline (nonpigmented) septated hyphae thus they may be confused with Aspergillus, Fusarium, and other septated molds as well as other yeast such as Trichosporon spp.25 In addition, Candida spp. can be observed with other fungal infections.26
The different fungal infections require different treatments and based on tissue morphology it is not possible to differentiate the genus of the groups mentioned. Special stains such as periodic acid Schiff and silver fungal stains (GMS) can be used to further highlight septations in the hyphae. Melanin stains such as Fontana-Masson can highlight pigment; however, stains for melanin can also be misleading as pigment can be observed in molds that are not dematiaceous or yeasts other than Cryptococcus which classically have a positive reaction.27 In addition, the use of commercial antibodies in immunohistochemical assays has proven to be disappointing for differentiation of the different fungi.28–30 Thus, application of ISH for rapid identification of fungal pathogens may be of benefit in situations where the speciation is critical for therapy. In addition, ISH may be useful in defining if there are infections by >1 fungus, or in cases for which fungal cultures are negative or have not been performed and a fungal pathogen is histologically observed.
IN SITU HYBRIDIZATION WITH DNA PROBES FOR FUNGAL rRNA SEQUENCES
The first fungal rRNA detection by ISH was carried out using DNA oligonucleotide probes.14–18,31–42 Subsequently, a variety of fungal pathogens have been detected in tissues by ISH with either biotin or digoxigenin probes including yeast (B. dermatitidis, C. immitis, Cryptococcus neoformans, H. capsulatum, and S. schenckii) and filamentous fungi (Mucorales, Pseudallsecheria bodyii, Fusarium sp, Candida sp. and dematiaceous fungi).31–42 ISH for fungi using DNA oligonucleotide probes show high degrees of specificity (often >90%) and various levels of sensitivity depending on the fungal agent being studied (ranging from 50% to 95%), can be performed in most instances in 1 to 3 hours, and have been used in a variety of settings including evaluation of invasive and noninvasive fungal rhinosinusitis, deep cutaneous fungal infections, pulmonary fungal infections, and for the evaluation of fatal culture negative disseminated fungal infections26,34–36,40 (Fig. 2).
LIMITATIONS OF IN SITU HYBRIDIZATION FOR rRNA SEQUENCES WITH DNA PROBES
There are multiple limitations for ISH for fungal organisms in tissue and cytologic substrates. First of all, signal can be weak if the rRNA is not abundant; however, most fungal rRNA is present in high copy numbers. With inadequately developed probes, signal may be weak or limited. Fuchs and colleagues constructed overlapping probes over the entire length of the Escherichia coli 16S and 23S rRNA sequences and they showed significant variability of ISH signals with different probes.43,44 In fact <25% of probes across the length of E. coli rRNA produced intense bright ISH signals and about 30% produced limited or no signal at all indicating that not all regions of rRNA sequences are available for hybridization. A similar study has not been performed to study fungal rRNA to the authors’ knowledge. The nature of rRNA is complex with the development of hairpin loops that are not always accessible. In addition, some areas of rRNA may be bound to proteins or other nucleic acids and not available for hybridization. rRNA sequences, particularly in fungi can have spatial localizations and therefore may not be available for hybridization in tissue preparations.45 An additional factor for fungal ISH is the variation in cell walls making enzymatic pretreatment and microwave pretreatment necessary steps for optimal signal development. Despite these methods, some fungal organisms still are difficult to produce consistently strong ISH signals. It is possible that ISH detects only viable organisms. In previous work, Park et al18 has shown that ISH for fungal pathogens is often weaker in entangled masses of fungi (fungus ball) and often show 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. Extensive tissue necrosis is often associated with reduced ISH signal.35,46 In order to increase the sensitivity of probes in light of these pitfalls with conventional ISH, a variety of other types of ISH probes have been developed including those with modified nucleotides.
IN SITU HYBRIDIZATION FOR FUNGAL DETECTION WITH MODIFIED NUCLEIC ACID PROBES
Locked Nucleic Acid Probes
Locked nucleic acids (LNA) are modified nucleotides in which the 2′ oxygen and the 4′ carbon are linked through a methylene unit.47–49 This change results in a “lock” on the nucleotide. LNA can be incorporated into RNA or DNA nucleic acid probes. These special nucleotides hybridize strongly to their complementary RNA and DNA, producing hybrids which are thermally stable.47–49 Because of these properties, probes of short length can be used and LNA probes can distinguish single mismatch and therefore can be useful for distinguishing between closely related species.
LNA probes are excellent for ISH procedures50–52 since LNA-DNA and LNA-RNA hybrids are much stronger than either RNA-DNA or RNA-RNA hybrids.47–49 The hybrids produced with LNA probes can withstand stringent conditions such as high temperatures (because of their thermal characteristics) and low salt concentrations.47–49 In fact, some studies have shown that LNA probes produce stronger ISH signals compared to the same sequence DNA probes as demonstrated in studies using LNA probes to detect microRNA sequences and rRNA sequences of some bacterial and fungal species,50–53 and this has been a justification for use of LNA probes despite the higher cost of synthesis. The number of studies that have used LNA probes for detecting rRNA sequences for microbiological speciation has been limited. Kubota et al were the first to use LNA probes to detect rRNA of bacteria by ISH.52 They observed an increased ISH signal with the addition of 2 to 4 LNAs per probe. Recent applications for fungal detection have included assays for Aspergillus sp., B. dermatitidis, H. capsulatum, C. immitis, Fusarium sp., Candida sp., and Mucorales genera54–58 (Fig. 3). Results have shown that hybridization signals are at least 2 times as strong compared to that seen with DNA probes of the same length and under the same stringencies.55–56
Peptide Nucleic Acid Probes
Peptide nucleic acid (PNA) is a nucleotide analog where a polyamide backbone replaces the traditional phosphate ribose ring of DNA. PNAs are capable of binding to DNA and RNA.59 In fact, PNA has superior binding to DNA and RNA and PNA-DNA and PNA-RNA complexes are stable. The PNA hybrids are resistant to nucleases and proteases and form independent of salt concentrations in solution.59 As a result, PNA probes are excellent tools for ISH. PNA probes have been of value for detecting rRNA of a variety of bacterial and fungal pathogens in blood culture and fluid specimens by fluorescence ISH,60–63 and methodologies using PNA probes for fungi in formalin-fixed, paraffin-embedded tissues have been recently reported64–66 (Fig. 4).
A proposed advantage of PNA probes is that due to their peptide-like qualities, these probes should be able to readily diffuse through cell walls negating the use of protease pretreatment. However, Montone and colleagues have shown that protease digestion remains necessary for optimal detection of the fungal rRNA sequences even with PNA probes.66–67
Applications of rRNA In Situ Hybridization in Diagnostic Medicine
To date utility of ISH for fungal rRNA sequences in medical practice has been limited but applications for these procedures are growing in tissue specimens. ISH using PNA, DNA, and LNA probes has been able to confirm suspected organisms in cases of noninvasive and invasive fungal sinusitis, deep cutaneous infections, solitary pulmonary nodules, and in disseminated fungal infections when cultures for the suspected pathogens were negative (Fig. 5). As more probes are developed, there will be likely further applications to these techniques.
rRNA sequences are abundant, conserved but species-specific sequences which make excellent target for ISH. Although culture is still considered the “gold standard” for diagnosing fungal infections, ISH may be useful when a pathogen is observed in tissue but cultures are negative, have not been performed, or there is suspicion of infection by >1 fungus.
1. Olsen GJ, Woese CR. Ribosomal RNA: a key to phylogeny. FASEB J. 1993;7:113–123
2. Olsen GJ, Woese CR, Overbeek R. The winds of (evolutionary) change: breathing new life into microbiology. J Bacteriol. 1994;176:1–6
3. Lane DJ, Pace B, Olsen GJ, et al. Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses. Proc Natl Acad Sci Oct. 1985;82:6955–6959
4. Stringer JR. The identity of Pneumocystis carinii
: not a single protozoan, but a diverse group of exotic fungi. Infect Agents Dis. 1993;2:109–117
5. Relman DA, Lepp PW, Sadler KN, et al. Phylogenetic relationships among the agent of bacillary angiomatosis, Bartonella bacilliformis
, and other alpha-proteobacteria. Mol Microbiol. 1992;6:1801–1807
6. Relman D, Schmidt ATM, MacDermott RP, et al. Identification of the uncultured bacillus of Whipple’s disease. N Engl J Med. 1992;327:293–301
7. Lane DJ, Stahl DA, Olsen GJ, et al. Phylogenetic analysis of the genera Thiobacillus
by 5S rRNA sequences. J Bacteriol. 1985;163:75–81
8. Olsen GJ, Pace NR, Nuell M, et al. Sequence of the 16S rRNA gene from the thermoacidophilic archaebacterium Sulfolobus solfataricus
and its evolutionary implications. J Mol Evol. 1985;22:301–307
9. Olsen GJ. Earliest phylogenetic branchings: comparing rRNA-based evolutionary trees inferred with various techniques. Cold Spring Harb Symp Quant Biol. 1987;52:825–837
10. Giovannoni SJ, DeLong EF, Olsen GJ, et al. Phylogenetic group-specific oligodeoxynucleotide probes for identification of single microbial cells. J Bacteriol. 1988;170:720–726
11. DeLong EF, Wickham GS, Pace NR. Phylogenetic stains: ribosomal RNA-based probes for the identification of single cells. Science. 1989;243:1360–1363 10
12. Lipski A, Friedrich U, Altendorf K. Applications of rRNA targeted oligonucleotide probes in biotechnology. Appl Microbiol Biotechnol. 2001;56:40–57
13. Burd EM. Validation of laboratory-developed molecular assays for infectious diseases. Clin Microbiol Rev. 2010;23:550–576
14. Montone KT. In situ hybridization for ribosomal RNA sequences: a rapid sensitive method for diagnosis of infectious pathogens in anatomic pathology substrates. Acta Histochem Cytochem. 1994;27:601–606
15. Hayashi Y, Watanabe J, Nakata K, et al. A novel diagnostic method of Pneumocystis carinii. In situ hybridization of ribosomal ribonucleic acid with biotinylated oligonucleotide probes. Lab Invest. 1990;63:576–580
16. Montone KT, Litzky LA. Rapid method for detection of Aspergillus
5S ribosomal RNA using a genus-specific oligonucleotide probe. Am J Clin Pathol. 1995;103:48–51
17. Kobayashi M, Urata T, Ikezoe T, et al. Simple detection of the 5S ribosomal RNA of Pneumocystis carinii using in situ hybridization. J Clin Pathol. 1996;49:712–716
18. Park CS, Kim J, Montone KT. Detection of Aspergillus
ribosomal RNA using biotinylated oligonucleotide probes. Diagn Mol Pathol. 1997;6:255–260
19. Lass-Flörl C, Mayr A, Perkhofer S, et al. Activities of antifungal agents against yeasts and filamentous fungi: assessment according to the methodology of the European Committee on Antimicrobial Susceptibility Testing. Antimicrob Agents Chemother. 2008;52:3637–3641
20. Guarner J, Brandt ME. Histopathologic diagnosis of fungal infections in the 21st century. Clin Microbiol Rev. 2011;24:247–280
21. Sangoi AR, Rogers WM, Longacre TA, et al. Challenges and pitfalls of morphologic identification of fungal infections in histologic and cytologic specimens. Am J Clin Pathol. 2009;131:364–375
22. Lee S, Yun NR, Kim K-H, et al. Discrepancy between histology and culture in filamentous fungal infections. Med Mycol. 2010;48:886–888
23. Patel AJ, Gattuso P, Reddy VB. Diagnosis of blastomycosis in surgical pathology and cytopathology: correlation with microbiologic culture. Am J Surg Pathol. 2010;34:256–261
24. Lemos LB, Guo M, Baliga M. Blastomycosis: organ involvement and etiologic diagnosis. A review of 123 patients from Mississippi. Ann Diagn Pathol. 2000;4:391–406
25. Luna MConnor DH, Chandler FW, Schwartz DA, et al. Candidiasis. Pathology of Infectious Diseases. 1997;Vol. 21st ed Hong Kong Stamford, Appleton & Lange Co:953–964
26. Louie C, Ende-Schwartz LB, Litzky LA, et al. Disseminated fungal infections at autopsy: detection of multiple fungal pathogens by in situ hybridization. Pathol Case Rev. 2011;16:260–265
27. Bishop JA, Nelson AM, Merz WG, et al. Evaluation of the detection of melanin by the Fontana-Masson silver stain in tissue with a wide range of organisms including Cryptococcus. Hum Pathol. 2012;43:898–903
28. Phillips P, Weiner MH. Invasive aspergillosis diagnosed by immunohistochemistry with monoclonal and polyclonal reagents. Hum Pathol. 1987;18:1015–1024
29. Reed JA, Hemann BA, Alexander JL, et al. Immunomycology: rapid and specific immunocytochemical identification of fungi in formalin-fixed, paraffin-embedded material. J Histochem Cytochem. 1993;41:1217–1221
30. Schuetz AN, Cohen C. Aspergillus
immunohistochemistry of culture-proven fungal tissue isolates shows high cross-reactivity. Appl Immunohistochem Mol Morphol. 2009;17:524–529
31. Hayden RT, Isotalo PA, Parrett T, et al. In situ hybridization for the differentiation of Aspergillus
, and Pseudallescheria
species in tissue section. Diagn Mol Pathol. 2003;12:21–26
32. Hayden RT, Qian X, Procop GW, et al. In situ hybridization for the identification of filamentous fungi in tissue section. Diagn Mol Pathol. 2002;11:119–126
33. Hayden RT, Qian X, Roberts GD, et al. In situ hybridization for the identification of yeastlike organisms in tissue section. Diagn Mol Pathol. 2001;10:15–23
34. Myoken Y, Sugata T, Mikami Y, et al. Identification of Aspergillus
species in oral tissue samples of patients with hematologic malignancies by in situ hybridization: a preliminary report. J Oral Maxillofac Surg. 2008;66:1905–1912
35. Montone KT, Livolsi VA, Feldman MD, et al. In situ hybridization for specific fungal organisms in acute invasive fungal rhinosinusitis. Am J Clin Pathol. 2011;134:190–199
36. Montone KT, LiVolsi VA, Feldman MD, et al. Rapid in situ hybridization for dematiaceous fungi using a broad-spectrum oligonucleotide DNA probe. Diagn Mol Pathol. 2011;20:180–183
37. Perez LA, Gupta PK, Montone KT. Detection of Pneumocystis Carinii in transbronchial biopsy and bronchoalveolar lavage specimens by in situ hybridization and immunohistochemical techniques. Cell Vision: J Anal Morphol. 1995;2:462–467
38. Perez-Jaffe LA, Lanza DC, Loevner LA, et al. In situ hybridization for Aspergillus
in allergic fungal sinusitis: a rapid means of speciating fungal pathogens in tissues. Laryngoscope. 1997;107:233–240
39. Zimmerman RL, Montone KT, Fogt F, et al. Ultra fast identification of Aspergillus
species in pulmonary cytology specimens by in situ hybridization. Int J Mol Med. 2000;5:427–429
40. Abbott JJ, Hamacher KL, Ahmed I. In situ hybridization in cutaneous deep fungal infections: a valuable diagnostic adjunct to fungal morphology and tissue cultures. J Cutan Pathol. 2006;33:426–432
41. Kobayashi M, Sonone H, Ikezoe T, et al. In situ detection of Aspergillus
18 S ribosomal RNA in invasive pulmonary aspergillosis. Intern Med. 1999;38:563–569
42. Shinozaki M, Okubo Y, Nakayama H, et al. Application of in situ hybridization to tissue sections for identification of molds causing invasive fungal infection. Jpn J Med Mycol. 2009;50:75–83
43. Fuchs BM, Wallner G, Beisker W, et al. Flow cytometric analysis of the in situ accessibility of Escherichia coli 16S rRNA for fluorescently labeled oligonucleotide probes. Appl Environ Microbiol. 1998;64:4973–4982
44. Fuchs BM, Syutsubo K, Ludwig W, et al. In situ accessibility of Escherichia coli 23S rRNA to fluorescently labeled oligonucleotide probes. Appl Environ Microbiol. 2001;67:961–968
45. Teertstra WR, Lugones LG, Wosten HAB. In situ hybridization in filamentous fungi using peptide nucleic acid probes. Fungal Genet Biol. 2004;41:1099–1103
46. Montone KT, Palmer J, Chiu AG, et al. In situ hybridization (ISH) for determination of fungal rRNA preservation in sinonasal fungal disease. Mod Pathol. 2010;23:436A
47. Kaur H, Wengel J, Maiti S Thermodynamics of DNA-RNA heteroduplex formation: effects of locked nucleic acid nucleotides incorporated into the DNA strand Biochemistry. 2008;47:1218–1227
48. Kurreck J, Wyszko E, Gillen C, et al. Design of antisense oligonucleotides stabilized by locked nucleic acids. Nucleic Acids Res. 2002;30:1911–1918
49. Koshkin AA, Nielsen P, Meldgaard M, et al. LNA (Locked Nucleic Acid): An RNA mimic forming exceedingly stable LNA:LNA duplexes. J Am Chem Soc. 1998;120:13252–13253
50. Amann R, Fuchs BM. Single-cell identification in microbial communities by improved fluorescence in situ hybridization techniques. Nat Rev Microbiol. 2008;6:339–348
51. Thomsen R, Nielsen PS, Jensen TH. Dramatically improved RNA in situ hybridization signals using LNA-modified probes. RNA. 2005;11:1745–1748
52. Kubota K, Ohashi A, Imachi H, et al. Improved in situ hybridization efficiency with locked-nucleic-acid-incorporated DNA probes. Appl Environ Microbiol. 2006;72:5311–5317
53. Kloosterman WP, Wienholds E, de Bruijn E, et al. In situ detection of miRNAs in animal embryos using LNA-modified oligonucleotide probes. Nat Methods. 2006;3:27–29
54. Montone KT. In situ hybridization for fungal pathogens using locked nucleic acid probes. FASEB J. 2008;22:708.4
55. Montone KT, Feldman MD. In situ detection of Aspergillus
ribosomal rRNA sequences using a locked nucleic acid (LNA) probe. Diagn Mol Pathol. 2009;18:239–242
56. Montone KT, Feldman MD, Peterman H, et al. In situ hybridization for Coccidioides immitis
5.8S ribosomal RNA sequences in formalin-fixed, paraffin-embedded pulmonary nodules using a locked nucleic acid (LNA) probe: a rapid means for speciation in tissue sections. Diagn Mol Pathol. 2010;19:99–104
57. Baliff J, Litzky L, Montone K. In situ hybridization for fungal pathogens in lung specimens using locked nucleic acid probes. Arch Pathol Lab Med. 2010;134:229–234
58. Montone KT. Differentiation of Fusarium
species by colorimetric in situ hybridization in formalin-fixed, paraffin-embedded tissue sections using dual fluorogenic-labeled LNA probes. Am J Clin Pathol. 2009;132:866–870
59. Paulasova P, Pellestor F. The peptide nucleic acids (PNAs): a new generation of probes for genetic and cytogenetic analyses. Ann Genet. 2004;47:349–358
60. Stender H. PNA FISH: an intelligent stain for rapid diagnosis of infectious diseases. Expert Rev Mol Diagn. 2003;3:649–655
61. Reller ME, Mallonee AB, Kwiatkowski NP, et al. Use of peptide nucleic acid-fluorescence in situ hybridization for definitive, rapid identification of five common Candida species. J Clin Microbiol. 2007;45:3802–3803
62. Drobniewski FA, More PG, Harris GS. Differentiation of Mycobacterium tuberculosis
complex and nontuberculous mycobacterial liquid cultures by using peptide nucleic acid-fluorescence in situ hybridization probes. J Clin Microbiol. 2000;38:444–447
63. Lefmann M, Schweickert B, Buchholz P, et al. Evaluation of peptide nucleic acid-fluorescence in situ hybridization for identification of clinically relevant mycobacteria in clinical specimens and tissue sections. J Clin Microbiol. 2006;44:3760–3767
64. Shinozaki M, Okubo Y, Nakayama H, et al. Application of in situ hybridization to tissue sections for identification of molds causing invasive fungal infection. Nihon Ishinkin Gakkai Zasshi. 2009;50:75–83
65. Shinozaki M, Okubo Y, Sasai D, et al. Identification of Fusarium
species in formalin-fixed and paraffin-embedded sections by in situ hybridization using peptide nucleic acid probes. J Clin Microbiol. 2011;49:808–813
66. Montone KT. Rapid in situ hybridization for fungal pathogens using a peptide nucleic acid (PNA) probe targeting a panfungal rRNA sequence. Mod Pathol. 2008;21(suppl 1):1671a
67. Montone KT, Feldman MD. Evaluation of DNA, LNA, and PNA probes for in situ detection of panfungal ribosomal RNA sequences in tissues: a multispectral imaging study. Mod Pathol. 2009;22(suppl 1):225a
fungus; in situ hybridization; ribosomal RNA; LNA; PNA
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