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Advances in Anatomic Pathology:
doi: 10.1097/PAP.0b013e31828d187d
Review Articles

In Situ Hybridization for rRNA Sequences in Anatomic Pathology Specimens, Applications for Fungal Pathogen Detection: A Review

Montone, Kathleen T. MD*; Guarner, Jeannette MD

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Author Information

*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: kmontone@upenn.edu).

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Abstract

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

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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.

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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.

Figure 1
Figure 1
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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.

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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).

Figure 2
Figure 2
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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.

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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

Figure 3
Figure 3
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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).

Figure 4
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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

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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.

Figure 5
Figure 5
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CONCLUSIONS

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.

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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

Keywords:

fungus; in situ hybridization; ribosomal RNA; LNA; PNA

© 2013 Lippincott Williams & Wilkins, Inc.

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