Culture-based methodologies are often considered to be the most accurate means of detecting infection in tissue specimens (1,21,26). Diagnosis of infections directly from histopathologic sections can be quite challenging, and is based on morphologic features, using both hematoxylin–eosin stain as well as more specialized stains (3,21). Detection of fungal organisms is generally directed against the wall of the organism, and most commonly includes methanemine silver and/or periodic acid–Schiff stains. The latter methods have several potential advantages with respect to culture. They are rapid, requiring only a few hours (occasionally overnight incubation is required), whereas culture can take from days to weeks. They maintain morphologic relationships with the surrounding tissue structure, and this is particularly beneficial when trying to determine whether an organism represents contamination, colonization, or true infection. Moreover, material is sometimes not sent for culture, or culture results may not be available immediately for the optimal care of the patient. Lastly, culture results can be confounded by recent therapy, and some organisms may not be able to be cultivated because of the use of inappropriate media/methods for a given organism, handling the clinical specimen before culture, or inherent limitations of currently available microbiologic techniques. In fact, in certain circumstances, the sensitivity of histopathologic examination has been shown to be greater than that of culture (19).
Despite these advantages, there are numerous potential drawbacks to relying on morphology for diagnosis of these pathogens. Overall, culture is generally considered more sensitive than histology. Organisms may be scant in number or stained poorly with conventional methods. When organisms are detected, identification to the species level can be difficult or impossible. The latter issues are of particular concern in the diagnosis of yeast or yeastlike elements in tissue sections. A number of different fungal organisms may produce such structures, and often overlap in size range and in other morphologic characteristics (3,21). Fungal organisms, including the agents of blastomycosis, coccidiomycosis, cryptococcosis, and histoplasmosis, can all be confused with one another in various circumstances. At the low end of the size range, Histoplasma capsulatum can be confused with microforms of Blastomyces dermatitidis, with endospores of Coccidioides immitis, and with species of Candida. Less commonly, Sporothrix schenckii, Penicillium marneffei, and Toxoplasma gondii enter into the differential diagnosis. Blastomyces may be mistaken for empty spherules of Coccidiodes, and Cryptococcus neoformans can look like Pneumocystis carinii or Histoplasma. There are a number of ways to help resolve these issues. Clinical history can be suggestive, and the surrounding tissue response, as seen on hematoxylin–eosin-stained sections may also be quite helpful (3,21,25). Additional special stains, particularly mucicarmine for the detection of Cryptococcus, have been used (3,21); and immunohistochemical techniques have been applied with a variable degree of success (4,6,18,20,21,24). Molecular diagnostic techniques, particularly in situ hybridization (ISH), have the potential to help arrive at a definitive diagnosis in these difficult cases.
Although ISH has found previously an important role in the diagnosis of viral infections in tissue (7,14), the detection and identification of fungal agents has been limited to studies involving candidiasis, aspergillosis, and pneumocystosis (8,9,11,12,16,17). ISH offers rapid turnaround, limited cost, and the potential for automation, together with a high degree of specificity. The sensitivity of these techniques, which was limited previously to the range of 10 to 50 copies of the target sequence (23), has been enhanced markedly by the use of various signal amplification methods that can detect just a few copies of the target sequence (2,5,22,23,28). The current study evaluates the use of ISH for the identification of yeastlike elements in tissue section, using fungal culture as the reference standard, and compares the results with morphologic identification in Grocott's methenamine silver (GMS)-stained slides.
MATERIALS AND METHODS
Clinical Specimens and Experimental Design
Deoxyribonucleic acid probes were designed to detect both the 18S and 28S ribosomal ribonucleic acid (RNA) sequences of five fungal organisms, including B. dermatitidis, C. immitis, C. neoformans, H. capsulatum, and S. schenckii. A “pan-fungal” probe, directed against a common 18S ribosomal RNA sequence for all five organisms, was also designed. A total of 128 formalin-fixed, paraffin-embedded tissue specimens were selected for evaluation from archival material at the Mayo Clinic, Rochester, MN, USA. Thirty of these cases were excluded from the study because they had too few organisms present (zero to two organisms seen on GMS-stained slides) to allow for consistent evaluation of other sections. Table 1 shows the remaining 98 specimens, stratified by tissue type. Each selected specimen had culture-proved positivity from the original operative specimen, for involvement by one of the five aforementioned organisms.
Results of histopathologic evaluation were based on original, archived surgical pathology reports. Duplicate hematoxylin–eosin-and GMS-stained tissue sections were cut and stained concurrent with and consecutive to sections taken for ISH. These duplicates were evaluated by one of us (R.T.H.) to ensure the continued presence of the diagnostic area of interest in each case. All tissue sections were cut at 4 μm. Hematoxylin–eosin and GMS staining was carried out using standard histologic laboratory methods (15).
Oligonucleotide probes specific for five different fungal species were designed from variable regions of the 18S and 28S ribosomal RNA genes (Table 2). Target sites were selected based on the analysis of sequence matches and mismatches BLAST (GenBank). The specificity of probes was checked using Genetic Computer Group software V10.1 (Madison, WI, USA). Probes showed no evidence of cross-reaction with sequences of bacteria, parasites, animals, or humans. As described previously (13), all probes were 3` tailed with digoxigenin-11-dUTP (Boehringer Mannheim, Indianapolis, IN, USA), then diluted to a final concentration of 2.0 ng/μL in hybridization buffer.
In Situ Hybridization
ISH was performed using a modified procedure, as described previously (13).
Pretreatment of Sections
Four-micrometer-thick paraffin sections were used for ISH. After deparaffinization and rehydration, sections were rinsed twice in diethyl pyrocarbonate-treated H20 for 2 minutes each. Endogenous alkaline phosphatase activity was quenched with 0.2 M HCl for 20 minutes at room temperature, and slides were microwaved for 10 minutes in 10 mM citric acid (pH, 6.0) and cooled to room temperature. Sections were then digested with 25 μg/mL proteinase K (Boehringer Mannheim) in 10 mM phosphate-buffered saline (pH, 7.2) for 10 minutes at room temperature, followed by acetylation for 15 minutes with freshly prepared 0.6% acetic anhydride in 0.1 M triethanolamine (pH, 8.0). Prehybridization was performed for 30 minutes at room temperature with a mixture containing 50% deionized formamide (Boehringer Mannheim), 10% dextran sulfate (Sigma), 1× Denhardt's solution (Sigma), 3× standard saline citrate, 100 μg/mL salmon sperm deoxyribonucleic acid (Sigma, St. Louis, MO, USA), 125 μg/mL yeast transfer RNA, 10 μg/mL polyadenylic–cytidylic acid, 0.05 M Tris, 5 mM ethylenediamine tetraacetic acid, 600 mM NaCl, and 0.1% inorganic sodium pyrophosphate.
Hybridization and Posthybridization Washes
After prehybridization, residual prehybridization buffer was removed thoroughly from around the tissue section. A pan-fungal oligonucleotide probe or a species-specific oligonucleotide probe cocktail (2 ng/μL in prehybridization buffer) was applied to sections. Slides were covered with a sigmacote™ (Sigma)-coated coverglass, heat treated at 95°C for 5 minutes, and hybridized in a humid environment for 3 hours at 50°C (overnight hybridization for tyramine signal amplification procedure). Sections were rinsed twice in 2× standard saline citrate for 10 minutes at room temperature, washed in 0.5× standard saline citrate at 37°C for 20 minutes (to remove excess probe), and rinsed twice in buffer A (1% normal sheep serum in 0.3% Triton X-100) for 2 minutes at room temperature.
Routine immunodetection: After posthybridization washing, digoxigenin-labeled probes were detected according to the manufacturer's instructions (digoxigenin detection kit; Boehringer Mannheim). Briefly, after preincubation of sections for 30 minutes in blocking buffer A (1% normal swine serum and 0.3% Triton X-100), the sections were incubated in a 1:200 dilution of alkaline phosphatase-conjugated antidigoxigenin Fab fragment, in blocking buffer A, for 1 hour at room temperature. Rinsing with buffer A and buffer C (Tris HCl and MgCl; pH, 9.5) was performed, and sections were subsequently reacted with nitroblue-tetrazolium chloride and 5-bromo-4-chloro-3-indolyphosphate, forming an insoluble blue precipitate at the site of reaction. Sections were then rinsed in buffer C, counterstained with 0.1% Nuclear Fast Red, rinsed again in buffer C, dehydrated in graded ethanols, cleared in xylene, and “coverslipped” with a xylene-based synthetic mounting medium.
Catalyzed Reporter Deposition (CARD): The CARD procedure (27), also known as tyramide signal amplification, was carried out on all specimens that were considered false negative by routine immunodetection (see Statistical Analysis). After posthybridization washing, the slides were washed three times with 2× standard saline citrate at room temperature (2 minutes each wash), and rinsed briefly in tris HCl-NaCl-tween (TNT) buffer (100 mM Tris HCl, 150 mM NaCl [pH, 7.5], containing 0.05% Tween-20). Immunoamplification was performed using the biotinyl indirect tyramide signal amplification reagent (NEN Life Science Products, Boston, MA, USA). After a 30-minute room temperature blocking incubation, slides were washed three times for 5 minutes in TNT, followed by incubation with anti-DIG-Fab-HRP (1:200 diluted in TNB blocking buffer provided in a kit). Sections were then washed three times, for 5 minutes each, in TNT wash buffer. Biotinyl–tyramide was diluted (1:50 in the diluent provided in the kit), and sections were incubated in the dark for 10 minutes at room temperature in this substrate. After three 5-minute washes in wash buffer, a 1:100 dilution of streptavidin–alkaline phosphatase conjugate (Gibco BRL Life Technologies, Gaithersburg, MD, USA) in TNB blocking buffer was applied to the sections for 1 hour at room temperature in the dark. Finally, sections were rinsed three times (5 minutes each) in wash buffer, followed by application of nitroblue–tetrazolium chloride/5-bromo-4-chloro-3-indolyphosphate as a substrate. Slides were counterstained with 0.1% Nuclear Fast Red, dehydrated through a graded ethanol series, and coverslipped.
ISH Negative Controls and Fungi Probe Specificity Test
Negative controls used for ISH consisted of 1) omission of the probes from the hybridization reaction, 2) slides hybridized with nonlabeled probe, 3) cross-reactivity tests for probe specificity, and 4) testing of false-negative cases with β-actin and poly-deoxy thymidine probes to demonstrate the presence of detectable nucleic acid. Cross-reactivity testing consisted of the application of each species-specific probe cocktail to 10 culture-proved cases of each of the other organism types tested (a total of 40 cases tested for each probe cocktail). In the case of the S. schenckii probe cocktail, only five cases of each of the other organisms (20 total cases) were tested for cross-reactivity.
Accuracy of the ISH procedure was assessed based on culture results, and was compared with the results of histopathologic staining with GMS. Sensitivity was defined as the ability to detect the presence of the same fungal organism as that identified by culture. Specificity was also based on the ability to identify correctly detected organisms to the species level, allowing an assessment of any cross-reactivity that might have occurred with species-specific ISH probes, and reflecting organisms that were misidentified by GMS (specificity = number of negatives detected for a given species/total number of true negatives). Results for sensitivity, specificity, and positive predictive value are given for B. dermatitidis, C. immitis, H. capsulatum, and C. neoformans. In the case of ISH, each species-specific probe was tested against all true positives for that organism, as well as against 10 known positive cases each of the other three organism types (30 true negatives tested for each probe). Calculations for sensitivity, specificity, and positive predictive value are given in Tables 3 and 4 using a 95% exact binomial confidence interval.
A total of 98 cases were included in the study (see Table 1), with the vast majority representing pulmonary specimens positive for B. dermatitidis, C. immitis, H. capsulatum, and C. neoformans. Because of limited sample availability, only four culture-proved cases of S. schenckii were analyzed. In all cases, at least two morphologically distinct organisms were seen on GMS staining and/or on treatment by ISH.
Histopathologic Examination of GMS-stained Slides
Examination of GMS-stained tissue sections revealed organisms in the vast majority of cases (Table 5). Looking at organism detection only (and not at correct organism identification), the only false negatives were recorded for H. capsulatum. Sensitivity, specificity, and positive predictive value of histologic examination, as described earlier, are shown in Table 3. Overall sensitivity of GMS stain for the study was 95.9%. However, on a species-specific basis, morphology proved somewhat less reliable, with sensitivities of 75.0 to 94.7% and specificities of 96.2 to 100%. A total of eight cases were misidentified (8.2% of all cases examined), in two of which no species diagnosis was made. In the other six cases, the wrong designation was made, in comparison with culture data from the same specimen. The largest number of cases in which an incorrect identification was made involved C. immitis; however, inaccuracies were also seen in cases of H. capsulatum, B. dermatitidis, and C. neoformans. The positive predictive value of silver staining (83.3–100%) also reflects the variable accuracy of this technique.
In Situ Hybridization
ISH results are summarized in Tables 4 and 5. Pan-fungal probes and species-specific probes had nearly complete concordance in terms of sensitivity. For all probe sets, positive organisms appeared blue (see Fig. 1) against an eosinophilic background in tissue sections. Although there was some variability in staining intensity and contrast, most cases were defined quite sharply, with a minimum of background staining. In particular, cases of C. immitis, B. dermatitidis, and C. neoformans were generally easily resolved, with even rare organisms readily identified. The most difficult organisms to identify were those of H. capsulatum, which were often absent or ill defined, particularly in cases that showed a marked degree of necrosis.
The sensitivity of ISH reflects these general observations. The three most easily seen organisms had sensitivities of 91.4 to 95.0%, whereas H. capsulatum was detected in only 50.0% of specimens. The trend toward reduced sensitivity for H. capsulatum was also seen, although to a lesser degree, in GMS-stained sections (see Table 3). Interestingly, four cases involving the latter organism were detected by ISH but not by silver staining. These were all bone marrow biopsies with a limited organism load, and no morphologically evident necrosis. All bone marrow biopsies entered in the study were positive by ISH, as were many of the cutaneous lesions. Most of these positive cases showed an absence of necrosis. Conversely, most of the false negatives were from pulmonary specimens, with variable numbers of organisms, but with abundant tissue necrosis. In fact, excluding cases with necrosis, the sensitivity of ISH for H. capsulatum increases from 50.0 to 87.5% (seven of eight cases).
Species-specific ISH probes appeared uniformly accurate, identifying correctly all organisms in which a signal was visualized by pan-fungal probes. Additional assays showed no evidence of cross-reactivity between each species-specific probe cocktail and representative cases of the other organisms tested in the study (100% specificity and 100% positive predictive value). For three of four organisms, ISH showed an advantage in terms of both specificity and positive predictive value. No advantage was demonstrated in the case of C. neoformans (100% specificity and positive predictive value for both methods). Because only four cases of S. schenckii were studied, no statistical evaluation of these results was performed.
Signal Enhancement by Catalyzed Reporter Deposition
The application of CARD had a beneficial impact on ISH sensitivity. CARD was used in all cases initially read as negative by ISH. Excluding Sporothrix cases, a total of 24 specimens were analyzed using this technique, 10 of which (41.7%) yielded a positive result. This represents 12.3% and 12.2% of all cases positive by pan-fungal and species-specific probes respectively. The impact was most dramatic for cases of C. immitis, B. dermatitidis, and C. neoformans. In the latter three categories, eight of 12 CARD-tested cases (66.7%) changed from a negative to a positive reading. For all cases in which CARD changed the results of ISH, it had a positive affect on the detection of both pan-fungal and species-specific hybridization. Although occasional cases showed increased background staining when compared with conventional ISH, in most cases this did not impair evaluation of the slides. All cases that were negative by CARD were tested with β-actin and poly-deoxy thymidine probes, demonstrating the continued presence of detectable nucleic acid in all but one case (one case of histoplasmosis was negative by both probes).
The current study demonstrates that oligonucleotide probes directed against ribosomal RNA sequences can be used for the rapid identification of yeast and yeastlike elements in formalin-fixed, paraffin-embedded tissue sections. Although examination of GMS-stained slides provided a high degree of overall sensitivity, ISH showed advantages in terms of accurately identifying fungal elements. This advantage may be particularly useful for cases in which culture data are delayed or otherwise unavailable. In a few cases, detection through the use of ISH was accomplished in the absence of organisms on silver-stained slides. This suggests that in cases with a high degree of clinical or histopathologic suspicion, the use of ISH can be justified, with the hopes of enhancing sensitivity beyond that achieved with conventional staining methods.
Conventional methods should remain as a first line of examination. Although the study design did not permit an overall assessment of sensitivity in comparison with culture, GMS staining clearly detected more cases of infection than ISH, with only four cases of histoplasmosis missed by GMS staining. There are inherent pitfalls in the sensitivity of silver staining, which include inconsistencies in stain quality, interobserver variability (i.e., false negatives resulting from stained organisms present but not seen on routine examination), and sampling error resulting from a paucity of organisms in the particular tissue block analyzed. The latter difficulties, however, can be found in the use of any histopathologic method (21). Specificity of GMS stain, as judged by the organism identity given on the final surgical pathology report, was somewhat less impressive than sensitivity. Misidentifications were made in each species category of culture-positive cases. There seemed to be particular difficulty in identifying correctly C. immitis, with an incorrect diagnosis in 11.4% of all cases involved by this species. This is not surprising because immature spherules can be quite variable in size, overlapping with nonbonding forms of both B. dermatitidis and C. neoformans in their appearance (3,21). Also, in some cases only endospores may be observed, suggesting the possibility of histoplasmosis or of small forms of other yeasts. Lastly, the occasional presence of hyphal structures can serve as an additional confounding factor in the correct identification of these organisms.
ISH was a useful tool in helping to make such species-level distinctions of various fungal organisms. Certainly the primary strength of ISH lies in its specificity and positive predictive value (100% in all cases when compared with culture). GMS staining interpretation showed variable results, often demonstrating reduced agreement with culture, in terms of organism identification. The ISH procedure, as described, is relatively easy to perform and provides easily interpretable results, with maintenance of both organism morphology and surrounding tissue architecture. The principal weakness of the procedure in this setting lies in its sensitivity. This was particularly troublesome with respect to cases of histoplasmosis, of which only 50% could be detected by ISH. It was not as problematic with other organisms, with the vast majority showing a strong hybridization signal. Likewise, the use of CARD, as a means of boosting sensitivity, enabled the visualization of only two additional cases of histoplasmosis. However, excluding cases of histoplasmosis and sporotrichosis, application of CARD picked up a majority of cases that were initially interpreted as negative by ISH.
The explanation for this relative lack of sensitivity for H. capsulatum is most likely multifactorial. A number of causes may explain a diminished effectiveness of ISH, including issues related to reagents, procedures, and specimens. One explanation in this case could be polymorphisms in various strains of the organism, reducing homology with the selected probes. This seems unlikely, because a cocktail of probes was used, representing both the 18S and the 28S ribosomal RNA sequences, and these sequences were highly conserved in all GenBank sequences examined. The small physical size of the organisms involved, and a correspondingly small amount of nucleic acid target may also be suggested. However, in this instance, the application of CARD would be expected to detect more positive cases. Variation in fixation and processing of the tissue specimens is doubtful as a primary etiology because other organisms did not show a corresponding variability in detection. The number of organisms present in the specimen did not appear to be a primary factor because there was no correlation between organism burden and detectability, either in the case of H. capsulatum or in the other organisms tested. More likely than the latter issues may be the presence of extensive necrosis in most of the cases of histoplasmosis that were undetected by ISH. The necrotic tissue may interfere with nucleic acid hybridization and may also prevent development of a recognizable signal. Additionally, extensive tissue degradation may result in the release of endogenous nucleases. Although the organism wall may remain detectable by GMS stain, its nucleic acid may be disrupted, preventing hybridization with the oligonucleotide probes. That tissue necrosis plays a role in the lack of sensitivity of ISH for this one organism group is further suggested by the much higher degree of sensitivity seen in bone marrow, skin, and other nonpulmonary specimens, all of which showed much less necrosis. Whatever the cause, this remains a major limitation of the use ISH technique to detect pulmonary H. capsulatum infection.
Despite this limitation, ISH appears to be a potentially valuable technique for specifically identifying yeastlike organisms in tissue sections. All five species-specific probe sets showed 100% specificity when checked for cross-reactivity with the other four organisms tested (an insufficient number of Sporothrix-positive cases were assayed to validate that probe set fully). Furthermore, results of the current study indicate that a substantial number of misidentifications may be avoided through the use of such a highly accurate tool as ISH. In other cases, when no attempt is made to identify specifically the organisms (“yeast forms present”), there could also be a benefit from the use of ISH.
This benefit stems not only from an increase in accuracy, with potential clinical implications in terms of treatment efficacy, but also from the timeliness with which such results may be made available. ISH can be performed with a 24-hour turnaround for a relatively limited cost. This contrasts with the fact that culture of these organisms can sometimes take several weeks (10). In addition, some cases may not be sent for culture, and in some cases submitted for culture, viable yeast may not be grown for identification purposes. The rapidity with which identifications can be made by ISH has potential implications for therapeutic decision making as well as for possible downstream costs and implications of an incorrect or deferred diagnosis. These may involve additional laboratory testing, inappropriate therapy, increased length of hospital stay, and potentially increased morbidity and mortality. Although GMS staining with morphologic identification is adequate in most cases, in large, referral-based settings, ISH may find a role in a number of selected cases on a routine basis.
Many such large reference centers already perform ISH for a number of purposes, related both to research and to pathology practice. In these cases, the additional availability of in situ probes for fungal elements can be made with a minimum of new capital investment, and without additional training of personnel. Furthermore, the successful use of ISH, as described in this report, suggests that probe sets may also be developed for other agents of fungal infection. Not only yeast, but also certain filamentous fungi often challenge the morphologist in attempts to label accurately the etiologic agent of infection. The application of a more comprehensive panel of ISH probes, in some cases enhanced through the use of signal amplification techniques, such as CARD, promises to improve our ability to identify unknown fungal pathogens quickly and accurately.
The results of the current study indicate that ISH may provide a rapid and accurate means of identifying yeast, and yeastlike fungal elements in difficult cases, when the paucity of organisms and/or atypical morphologic features prevent a definitive identification from GMS-stained tissue sections. Traditional morphologic evaluation remains essential, and has clear advantages in terms of its sensitivity for the detection of such infective agents. However, the adjunctive use of ISH, in some cases combined with other signal amplification methods, can provide an increased specificity, with added advantages of rapid turnaround and low technical costs. These features suggest that ISH can provide clinically important information, which may not be readily available through the use of current methods.
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Keywords:© 2001 Lippincott Williams & Wilkins, Inc.
In situ hybridization; Yeast; Ribosomal RNA; Histopathology