A Comparison of Histological Staining Methods for Pathogenic Fungi in Humans

: Background: Using special stains to detect fungus is an essential diagnostic tool in histology. Because microbiology cultures can take over a month for a definitive diagnosis, rapid detection and treatment of fungal infections are necessary for patient care, particularly in immunocompromised patients when infections may lead to life-threatening complications. Objective: A comparison of various classic special stain methods along with more recent modifications on those staining methods was performed to evaluate the effectiveness of fungal detection while attempting to ameliorate the dangers associated with certain chemicals used in histochemical procedures. Methods: Twenty-five formalin-fixed paraffin-embedded archived tissue blocks containing the genera Aspergillus, Blastomyces, Candida, Cryptococcus, Histoplasma, Mucor


Introduction
Although fungal elements are classified under the kingdom Fungi, there are multiple subkingdoms/phyla due to differences in organismal structure, life cycle, growth pattern, host preference, and physiological requirements.The variations in cell wall composition preclude most staining protocols from achieving 100% sensitivity in visualizing all pathological fungal species [1].In addition, differences in fixation times and tissue processing protocols may inhibit or enhance detection of fungal elements depending on the staining method used.Rapid detection and accurate identification of pathogenic fungi in human tissues are critical for effective treatment, and rapid techniques for frozen sections as well as paraffin-embedded sections have been developed [2,3].Commonly identified pathological fungi found in the Southeast United States include Histoplasma capsulatum, Candida spp., Pneumocystis jirovecii, Blastomyces dermatitidis, Cryptococcus neoformans, and Aspergillus spp.
Most aldehyde-based fungal stains such as the Hotchkiss-McManus method [4], also known as periodic acid Schiff light green stain (PAS-LG), are somewhat selective.While many fungi are highlighted well, the Schiff reagent occasionally labels hyphae, pseudo-hyphae, and yeast forms poorly or produces a similar-colored background which conceals morphology of the organism [5].The Gridley stain [6] is also an aldehyde-based stain but uses chromic acid and paraldehyde which are both considered health hazards and undesirable in the routine laboratory.Gomori [7] and Grocott [5] silver protocols produce mixed staining results on fungal genera based on oxidizer and impregnation solution used.Chromic acid is the traditional oxidizer for silver stains and produces excellent labeling of most fungal organisms [8].However, chromic acid is also very corrosive, toxic, and requires special disposal considerations [9].For those reasons, chromic acid is considered dangerous for routine use and has been removed from most clinical pathology laboratories.Previous investigators have attempted to identify variations in the Grocott method which reduce safety hazards but obtain similar results.Periodic acid has replaced chromic acid in most laboratories where automated methods or manual kits are used, resulting in reduced safety concerns but also in reduced sensitivity to fungal labeling [8].Heating the tissue sections prior to oxidizing with periodic acid [10] or heating tissue sections in periodic acid [8] are methods suggested in the literature to increase organism visualization.While histoplasma is one of the most common fungi found in surgical resections, it is also one of the most variably staining fungi [8].To date, commercially available antibodies to histoplasma that can be used with immunohistochemistry (IHC) procedures are scarce.However, Ku et al. found that an antibody to platelet and megakaryocyte CD42b receptors also labeled histoplasma organisms [11].The purpose of this study was to compare various commonly used as well as some lesser-known staining protocols, with and without modifications, to compare staining effectiveness on pathogenic fungi in humans.
For all staining protocols indicated, tissue sections were first deparaffinized through 3 changes of xylene, 1 minute each, and hydrated through 2 changes of absolute ethanol, 1 change of 95% ethanol, and de-ionized water (DIH 2 O), 10 dips each.After completion of each staining protocol, tissue sections were dehydrated through 3 changes of absolute ethanol, 10 dips each, and immersed in 3 changes of xylene, 1 minute each, prior to coverslipping using a resinous mounting medium.Staining protocols were all manually performed and included hematoxylin and eosin (H&E) (Table 1), periodic acid Schiff with light green or metanil yellow counterstains (Tables 2&3), chromic acid Schiff with metanil yellow counterstain (Table 4), Gomori method (Table 5), 4% periodic acid with Gomori reagent (Table 6), 4% periodic acid at room temperature (RT) and heated to 60°C followed by methenamine silver (Table 7), 1% potassium permanganate methenamine silver (Table 8), 1% periodic acid at RT and heated to 60°C followed by methenamine silver (Table 9), Grocott method (Table 10), and gram stain (Table 11).In addition, the investigators attempted heat pretreatment of tissue sections with deionized water at 120°C for 5 minutes in a decloaking device and at 60°C for 1 hour in a water bath prior to the 1% Gomori method for fungal demonstration (1% PAG) procedure in the attempt to increase positive labeling of fungal elements.In addition to special stain methods implemented, manual immunohistochemistry using a high pH epitope retrieval solution in a decloaking chamber at 120°C for 5 minutes followed by IHC labeling using a Bond™ Polymer Refine Detection Kit (DS9800, Leica Biosystems, USA) and 2 mouse monoclonal antibodies to CD42b (sc-59052 and sc-271171, Santa Cruz Biotechnology, USA) were employed at dilutions of 1:75 and 1:150, respectively, in the attempt to identify histoplasma organisms.
All tissue sections were evaluated independently by both investigators using a grading scale of 0-3 where 0 indicates no staining of organism, 1 = mild staining of organism but unacceptable for routine detection purposes, 2 = moderate but not optimal staining of organism, acceptable for purposes of detection, and 3 = optimal staining of organism.Additionally, the investigators noted whether the background stain made visualization of the fungi difficult.Discrepancies between the scores were resolved together at the microscope.Images were obtained using a 100x objective on an Olympus BX45 light microscope (Olympus Corp, Japan) and CellSens software (Olympus).

Results
Staining results demonstrate a wide range of sensitivities (Table 12, Figure 1).Candida (Figure 2) and Cryptococcus were the most readily identifiable genera.Traditional staining protocols PAS-LG, 1% PAG, and 5% CAMS identified all 5 Candida and Cryptococcus organisms with the exception of PAS-LG only faintly labeling one Cryptococcus sample.Aspergillus was less-easily stained with only the 4% PAG, 4% PAMS, and 5% CAMS sufficiently marking all 3 samples.The 2 Blastomyces specimens (Figure 3) were adequately depicted in approximately half the staining protocols, all of which were silver impregnation methods rather than aldehyde methods.Pneumocystis carinii (Figure 4) organisms are found in a foamy or frothy appearing matrix produced as a cast in alveoli.Although many of the fungal stains adequately identified this foamy carbohydrate matrix, only 4% PAMS, 1% PAMS with oxidation carried out at 60°C for one hour, and 5% CAMS labeled the actual organisms.Histoplasma capsulatum (Figure 5) organisms were highlighted by most stains for fungal identification very poorly.Only the silver stain with 5% chromic acid as the oxidizer (5% CAMS) adequately identified all histoplasma organisms.Efforts to label histoplasma organisms with antibodies to CD42b using IHC were unsuccessful.However, both CD42 antibodies labeled platelet and megakaryocyte tissue controls in bone marrow very well.The single Mucor (Figure 6) specimen available was labeled by 1% PAS-LG, 1% PAG, 4% PAG, 4% PAMS, and 1% KMnO 4 with the latter showing the best contrast to the surrounding tissues.Surprisingly, the 5% CAMS protocol failed to label this single specimen adequately.The most sensitive staining method overall with 96% sensitivity was the original Gomori method using a 1-hour 4% chromic acid oxidation and methenamine silver impregnating reagent.Tissue sections preheated in DIH 2 O prior to undergoing 1% PAMS protocol demonstrated an unacceptable tissue loss of 44% (11 of 25 cases) due to tissues dislodging from microscope slides.Both the gram stain and the 5% chromic acid Schiff with metanil yellow counterstain (5% CAS-M) were the least successful protocols, each with 16% sensitivity overall and only adequately labeled Candida and Cryptococcus genera.

Figure 1. Overall percentage of organismal labeling for each staining method. PAS-LG -periodic acid Schiff with light green counterstain, PAS-M -periodic acid Schiff with metanil yellow counterstain, CAS-M -chromic acid Schiff with metanil yellow counterstain, PAG -periodic acid with ready-to-use Gomori methenamine silver solution, PAMS -periodic acid with in-house
prepared methenamine silver solution, KMnO4 -potassium permanganate followed by inhouse prepared methenamine silver solution, CAMS -chromic acid followed by in-house prepared methenamine silver solution.H&E -hematoxylin and eosin, PAS-LG -periodic acid Schiff with light green counterstain, PAS-M -periodic acid Schiff with metanil yellow counterstain, CAS-M -chromic acid Schiff with metanil yellow counterstain, PAG -periodic acid with ready-to-use Gomori methenamine silver solution, PAMS -periodic acid with in-house prepared methenamine silver solution, KMnO4 -potassium permanganate followed by in-house prepared methenamine silver solution, CAMS -chromic acid followed by in-house prepared methenamine silver solution.*Foam cast material of Pneumocystis seen, not organisms.**Heat pretreatment consisted of sections heated to either 120°C for 5 minutes in a decloaking device or at 60°C for 1 hour

Discussion
The PAS-LG stain represents the most used fungal stain on dermatological specimens in the Tennessee valley region because appreciable background staining is not often a problem in the epidermis.PAS-M is the same fungal stain, but with a metanil yellow background rather than light green.The color combinations produced by these two counterstains as they pair with Schiff reagent do not make fungal detection statistically different (56% sensitivity with PAS-LG and 60% sensitivity with PAS-M) and counterstain applied is essentially a matter of pathologist preference.
The 1% PAG stain represents the most commonly used silver stain in larger hospital laboratories with automated staining kits.The oxidizer is 1% periodic acid, and the silver impregnation solution is a pre-made methenamine silver.These kit reagents are convenient to use but have a relatively short shelf life.In this study, the 1% PAG adequately labeled approximately half (56%) of the fungi tested and performed particularly poorly on samples containing Aspergillus and Histoplasma, consistent with results from a previous study [8].One of the variables employed in comparing staining protocols was using a heat pretreatment of tissue sections prior to performing a silver stain with a periodic acid oxidizer.Although previous investigators have reported successful enhancement of fungus visualization using various solutions, heating methods, and times [10], the current investigatory team was unable to duplicate that work.When using heat pretreatment with deionized water at 120°C for 5 minutes in a decloaking device or 60°C at 1 hour in a water bath, tissue section loss from the microscope slide was unacceptably high at 44% (11 of 25 sections lost).Presumably, the previous study investigators used slides with increased adhesive properties, or possibly the tissue processing protocol was different.For the sections retained on the glass slides in the current study, comparable staining was noted to those samples undergoing the same staining protocol without heat pretreatment.Another variation of periodic acid/silver staining put forward by Carson et al. to increase oxidation was to incubate the tissue sections in 1% periodic acid at 60°C for one hour [8].The current study found that only 44% of tissue sections stained adequately using this method.
The investigators attempted to increase the oxidative capacity of the 1% PAMS stain by increasing the concentration of periodic acid from 1% to 4% and, like the Grocott method, allowing oxidation to occur at RT for 1 hour.This method increased acceptable staining results from 56% to 76%.The same protocol using 1% periodic acid incubated for 1 hour at RT resulted in 64% acceptability when it was paired with a freshly prepared methenamine silver solution rather than a commercially prepared solution.Further experimentation revealed that incubation of the tissues in 4% periodic acid for 1 hour at 60°C resulted in a significant drop to 24% sensitivity from the traditional stain which evidenced 56% sensitivity.
The 5% CAMS (chromic acid methenamine silver) is the traditional Grocott stain using chromic acid oxidation at room temperature for 1 hour followed by impregnation by methenamine silver solution at 60°C and was the most effective method in identifying fungal components with 96% sensitivity.The only organism poorly labeled was that of the one Mucor sample.Although this method is very effective, there are two distinct disadvantages to using the 5% CAMS protocol.First, chromic acid is toxic and corrosive and requires special disposal considerations [9,10].Secondly, this method produces occasional background staining (Figures 3h and 6h) seen in 24% of the samples evaluated in this study.The background staining is due to the non-specific interaction of the silver nitrate solution with tissue elements such as glycogen, elastin, and mucin [5] and can, on occasion, provide poor contrast to the organisms.The evaluator must then rely on discerning the shape of the organism only as opposed to employing both shape and color contrast to detect fungi.Background staining was, however, not specific to the 5% CAMS method, and the 1% PAS-LG, 1% PAS-M, and 4% PAMS protocols also produced background staining in a comparable percentage of specimens.
Although several protocols labeled Mucor acceptably, the only ones to produce a score of 3 were PAS-LG and 1% KMnO 4 .The optimal labeling of fungal components by KMnO 4 was achieved by the combination of a 0.5% potassium permanganate solution for 5 minutes at room temperature.The use of higher concentrations (>1%) of potassium permanganate as the oxidizing reagent resulted in pale labeling of fungal organisms.Subjecting tissue sections to longer incubation times (30 minutes) resulted in tissue loss.Unfortunately, because there was only one Mucor specimen available for evaluation, it is uncertain whether specifically the 1% KMnO 4 procedure would be reproducible on other Mucor specimens, and so these results must be interpreted with caution.
While the gram stain labels most bacterial species as gram positive or gram negative based on components in the cell membrane, it has the capacity to also label some fungal samples.Most notably, it is reliable in staining Candida, and usually Cryptococcus, but did not label any of the other genera of fungi in this study.Having a 16% sensitivity rate makes this stain a poor option for fungal detection.The Gridley stain is another stain noted in the literature [6] that makes use of chromic acid as the oxidizer and Schiff reagent to label but also requires aldehyde-fuchsin to enhance marking of fungal organisms.Paraldehyde is a controlled substance and is not available for routine use.The investigators attempted a modified version of this stain by omitting the paraldehyde (5% CAS-M) but had very little success detecting organisms with 16% sensitivity.
With Pneumocystis specifically, most methods performed in this study allowed visualization of the cast material only associated with the organisms.While the cast material may be considered by some pathologists as morphologically diagnostic [12], only a few of the silver stains including 5% CAMS and 4% PAMS revealed the actual organisms.
The incidence of Histoplasma is on the rise in the Americas [13,14] with the increase in immunocompromised persons making up the bulk of symptomatic cases.Although human immunodeficiency virus provided the impetus in cases of histoplasmosis in the 1980s, immunosuppression is increasingly found in diabetics, in persons who have undergone solid organ or bone marrow transplant, in persons being treated for cancer, and in persons with autoimmune disorders [14,15].Patient outcomes are significantly improved with rapid detection and treatment [15].Diagnostic testing for histoplasmosis is most frequently performed on blood or urine while special stains are used to evaluate surgical samples with varying sensitivity.To date, there is no widely available antibody to detect Histoplasma organisms using IHC on surgical tissue samples.The current study found that although the 2 antibodies used against CD42b antigenic site highlighted megakaryocytes admirably, there was no concurrent labeling of Histoplasma organisms.However, the antibodies employed in the current study were not the same clone as that used by Ku et al. [11], and it may be that their clone exhibited some cross-reactivity with the Histoplasma organism.

Conclusion
Many investigatory teams have attempted to improve special stain protocols for fungal element visualization while reducing the exposure of laboratory personnel to hazardous chemicals.While many protocols have met with success in these aspects, the current investigation suggests that the use of a 5% chromic acid oxidizer paired with a freshly prepared methenamine solution produces the highest sensitivity in fungus detection despite the occasional labeling of background tissue elements.

Study Approval
This study was approved by the University of Tennessee Health Science Center Internal Review Board number 22-08709-NHSR.

Table 11 .
Gram Stain for Bacteria.

Table 12 .
Acceptable staining results given as a percentage for each fungus type as compared with staining protocols.