Chapter 27 Special Stains Use in Fungal Infections

Transcription

1 Chapter 27 Abida Haque, MD Histologic evaluation of tissues is a quick and easy way to identify fungal organisms, and a strong adjunct to microbiologic culture for diagnosis of fungal infections. Histologic evaluation of granulomatous inflammation and granulomas must include special stains to exclude or include presence of fungi and acid-fast bacteria. Gomori Methenamine Silver (GMS) and Periodic acid-schiff (PAS) are the two most common stains used to look for fungi in tissues and in cytology specimens in the daily practice of pathology. The presence of fungus in the tissue sections provides an indisputable evidence of invasive infection. Because of their size and morphologic diversity, many fungi can be seen in tissue sections by conventional light microscopic examination of Hematoxylin and Eosin (H&E) stained sections. In cytology specimens, fungi can be identified by their size and specific morphology. In the tissues, fungi usually occur either as hyphae, budding yeast, endosporulating spherules, or a combination of these forms (1, 2). In some groups of fungi only one species of fungus is the cause of mycosis, and therefore when classic forms are observed, an etiologic diagnosis can be made. These groups of fungal diseases include adiaspiromycosis, blastomycosis, coccidioidomycosis, cryptococcosis, Histoplasmosis capsulati, Histoplasmosis duboisii, paracoccidioidomycosis, Penicilliosis marneffei, protothecosis, rhinosporidiosis, and sporotrichosis. Other mycoses are caused by any of the several species of a genus, all of which are morphologically similar in tissue sections. Although these fungi cannot be identified as to the species by conventional histology, the disease that they cause can be diagnosed generically; for example, aspergillosis, candidiasis, and trichosporonosis. Still other mycoses are caused by any of a number of fungi belonging to different genera. These fungi appear similar, if not identical to one another in tissues. With these fungi, it is not possible to identify the etiologic agent, however, the mycosis can be named; for example, phaeohyphomycosis and zygomycosis. Hematoxylin & Eosin is a versatile stain that enables the pathologist to evaluate the host response, including the Splendore-Hoeppli phenomenon, and to detect other micro-organisms (3). It is the stain of choice to confirm the presence of naturally pigmented fungi, and to demonstrate the nuclei of yeast-like cells. However, there are drawbacks to using just the H&E stain for fungal diagnosis. It is often difficult to distinguish poorly stained fungi from tissue components, even at higher magnifications. When sparse, fungi are easily overlooked in H&E stained sections. The morphologic features may not be evident and sometimes may be misleading. For example, Histoplasma, Blastomyces, and Paracoccidiodes may have cytoplasmic retraction artifact in the sections, making morphologic evaluation difficult. Some of the fungal variants may have different sizes, such as the large form variant (African) histoplasma, and microform blastomycosis. Some of the dimorphic fungi can form pseudohyphae in tissues. Sometimes the fungal morphology may be altered by therapy. Special stains for fungi are therefore essential for histopathologic evaluation of unexplained inflammatory processes (4, 5). Most fungi can be readily demonstrated with the common special stains, Gomori s methenamine silver (GMS), Gridley s fungus (GF), and periodic acid-schiff (PAS), also referred to as broad spectrum fungal stains. GMS is preferred for screening, because it gives better contrast, and stains even degenerated and nonviable fungi that are sometimes refractory to the other two stains (Fig. 1, 2). GMS also stains algae (Prototheca and Chlorella spp.), cyst walls of Pneumocystis jiroveci (Fig. 3, 4), pathogenic free living soil amebas, the spore coat of most microsporidian parasites, intracytoplasmic granular inclusions of Cytomyeolovirus, Actinomyces Israeli and related species, Nocardia spp., most Mycobacterium spp., and nonfilamentous bacteria with polysaccharide capsules such as Klebsiella pneumoniae and Streptococcus pneumoniae. Prolonged staining in the silver nitrate solution may be required to adequately demonstrate degenerated fungal elements such as the yeast-like cells of Histoplasma capsulatum var. capsulatum in granulomas. The disadvantage of GMS and GF fungal stains is that they mask the natural color of pigmented fungi, making it impossible to determine whether a fungus is colorless hyaline or dematiaceous (pigmented). Such a determination is crucial in the histologic diagnosis of mycosis caused by dematiaceous fungi such as phaeohyphomycosis (6). Except for the PAS reaction, fungal stains GMS and GF do not special stains and H & E 233

2 Figure 1. Hematoxylin and Eosin staining of Aspergillus. Figure 3. Hematoxylin and Eosin staining of Pneumocystis jirovici. Figure 2. GMS staining of Aspergillus. Figure 4. GMS staining of Pneumocystis jirovici. 234 special stains and h & E special stains and H & E 235

3 Figure 5. Alcian Blue staining of Cryptococcus. Figure 7. Hematoxylin and Eosin staining of Malassezia. Figure 6. PAS staining of Cryptococcus. Figure 8. PAS staining of Malassezia. 236 special stains and h & E special stains and H & E 237

4 Figure 9. Hematoxylin and Eosin staining of Histoplasma. Figure 10. AFB staining of Histoplasma. adequately demonstrate the inflammatory response to fungal invasion. To counteract this, a GMS-stained section can be counterstained with H&E for a simultaneous study of the fungus and the host response. The PAS stain performs almost as well as GMS, in screening for fungi. It actually demonstrates fungal morphology better than the silver stains. PAS can stain degenerated fungi that may not be visible on H&E stain. Calcific bodies that are sometimes found in caseating granulomas are also stained with PAS, and can be mistaken for yeast-like fungi. This is especially true when calcific bodies are apposed to give the false impression of budding yeasts, or when the bodies are laminated to give the appearance of a capsule or thick cell wall. Best stains to avoid this misinterpretation are GMS and GF stains, because the chromic acid used as an oxidizer in these stains dissolves the calcium, leaving the calcific bodies unstained. Conversely, there are artifacts that mimic fungi on GMS and GF stains that are not seen on PAS stain, therefore the use of both silver and PAS can reduce the incidence of false positive results. Narrow-Spectrum Fungal Stains The differential diagnosis of fungi may require the use of additional special stains that stain some fungal organism and not others. These are sometimes referred to as narrow-spectrum fungal stains (7, 8). Some of the stains in this category are mucin stains such as alcian blue and Mayer s, or Southgate s mucicarmine, that readily demonstrate the mucoid capsule of Cryptococcus neoformans (Fig. 5, 6). This staining reaction differentiates Cryptococcus from other fungi of similar morphology, such as Coccidiodes, Candida, and Histoplasma. These mucin stains are not specific for C. neoformans; the cell walls of B. dermatitidis and Rhinosporidium seeberi are often stained to varying degrees with mucin stains. However, these two fungi are nonencapsulated and morphologically distinct, and not ordinarily mistaken for Cryptococcus. In some cases, poorly encapsulated cryptococci in tissue sections may not stain positive with mucicarmine stain. In these cases, since the cell wall of C. neoformans contains silver reducing substances, possibly melanin precursors, it can be stained with Fontana-Masson s silver procedure for melanin (9, 10). This stain is especially useful in those cases of Cryptococcosis with invasive yeast forms that do not have readily detectable capsules, the so-called dry variants. Such forms could possibly be confused with non-encapsulated yeasts of similar morphology. Fontana-Masson and Lillie s ferrous iron stains for melanin can also be used to confirm and accentuate the presence of melanin or melanin-like pigments in the cell walls of poorly pigmented agents of phaeohyphomycosis in tissue sections (11). PAS may be used as a narrow-spectrum fungus stain. For example, in the differential diagnosis of small budding yeast forms, a weak PAS and a strong GMS staining favors a diagnosis of Histoplasma, since Candida, microforms of Blastomyces, and yeast forms of Malassezia show a strong cell wall staining with PAS (Fig. 7, 8). Another narrow-spectrum fungus stain is Ziehl-Neelson (ZN). In one study, 60% of blastomyces and 47% of histoplasma organism showed positive cytoplasmic staining of the yeast-like cells with ZN stain (Fig. 9, 10). No staining was seen in Cryptococcus or Candida, and very rare acidfast staining was seen in coccidiodomyces endospores (12). However, these staining properties are inconsistent and should not be used for primary diagnosis. The cell walls of fungi are in general, not acid fast. Autoflourescent Fungi Some fungi or fungal components in the H&E stained tissue sections are autofluorescent when examined under ultraviolet light source (13). Candida species, Coccidioides immitis and Aspergillus species can exhibit bright green to yellow-green autofluorescence (14). When sections of these fungi are stained with the PAS, bright yellow fungal autofluorescence against a deep red-orange background is seen (15). Autofluorescence may help delineate sparse or poorly stained fungi in H&E stained sections, however this property is inconsistent and should not be used for definitive diagnosis. Most fungi in frozen or paraffin embedded tissue sections also stain nonspecifically with Calcofluor white, a cotton whitener that fluoresces under ultraviolet light (16). This rapid and a simple fluorescence procedure can be routinely used in the intraoperative examination of fresh-frozen tissues for fungi. Immunoperoxidase stains can be used to identify certain fungi in smears and in formalin fixed, paraffin embedded tissue section. This technique, however, has only limited diagnostic use (17). 238 special stains and h & E special stains and H & E 239

5 Direct Immunofluoresence (IF) Direct immunofluoresence (IF) can improve the diagnostic capability of conventional histopathology in the diagnosis of fungal diseases (18). The IF procedure, which can be performed on smears and on formalin fixed paraffin embedded tissue sections is helpful in confirming a presumptive histologic diagnosis, especially when fresh tissues are not available for culture or when atypical fungus forms are seen. The Division of Mycotic Diseases, Center for Disease Control, Atlanta (United States) and others have a broad battery of sensitive and specific reagents available for identifying the more common pathogenic fungi. The immunofluoresence procedure has several advantages. Final identification of an unknown fungus is possible within hours after H&E and GMS stained sections are initially examined. The need for time consuming and costly cultures is often obviated by IF, and the hazards of handling potentially infectious materials are reduced when microorganisms are inactivated by formalin prior to IF staining. Prolonged storage of formalin fixed tissues, either wet tissue or paraffin embedded, does not appear to affect the antigenecity of fungi. This antigenic stability makes possible retrospective studies of paraffin embedded tissue and the shipment of specimens to distant reference laboratories for confirmatory identification. Most service laboratories, however, do not routinely use IF, since the special stains have been very reliable for diagnosis in the day to day pathology practice. References 1. Chandler FW, Watts JC. Pathologic Diagnosis of Fungal Infections Chicago: ASCP Press; Haque AK, McGinnis MR. Chapter 10. Dail and Hammar s Pulmonary Pathology New York: Springer; Liber AF, Choi HS. Splendore-Hoeppli phenomenon about silk sutures in tissue. Arch Pathol Lab Med 1973; 95: Schwartz J. The diagnosis of deep mycoses by morphologic methods. Hum Pathol 1982; 13: Vacca LL. Laboratory Manual of Histochemistry. New York: Raven; Chandler FW, Kaplan W, Ajello L. Color Atlas and Text of the Histopathology of Mycotic Disease. Chicago: Year Book Medical Youngberg GA, Wallen EDB, Giorgadze TA. Narrow-Spectrum histochemical staining of fungi. Arch Pathol Lab Med 2003; 127: Hussain Z, Martin A, Youngberg GA. Blastmyces dermatitides with large yeast forms. Arch Pathol Lab Med 2001; 125: Kwon-Chung KJ, Hill WB, Bennett E. New, special stain for histopathological diagnosis of cryptococcosis. J Clin Microbiol 1981; 13: Lazcano O, Speights VO Jr, Stickler JG, Bilbao JE, Becker J, Diaz J. Combined histochemical stains in the differential diagnosis of Cryptococcus neoformans. Mod Pathol 1993; 6: Wood C, Russel-Bell B. Characterization of pigmented fungi by melanin staining. Am J Dermatopathol 1983; 5: Wages DS, Wear DJ. Acid-fastness of fungi in blastomycosis and histoplasmosis. Arch Pathol Lab Med 1982; 106: Mann JL. Autofluorescence of fungi: an aid to detection in tissue sections. Am J Clin Pathol 1983; 79: Graham AR. Fungal autofluorescence with ultraviolet illumination. Am J Clin Pathol 1983; 79: Jackson JA, Kaplan W, Kaufman L. Development of fluorescentantibody reagents for demonstration of Pseudallescheria boydii in tissue. J Clin Microbiol 1983; 18: Monheit JE, Cowan DF, Moore DG. Rapid detection of fungi in tissue using calcofluor white and fluorescence microscopy. Arch Pathol Lab Med 1984; 108: Kobayashi M, Kotani S, Fujishita M, et al. Immunohistochemical identification of Trichosporon beigelii in histologic sections by Immunoperoxidase method. Am J Clin Pathol 1988; 89: Reiss E, de-repentgny L, Kuykendall J, et al. Monoclonal antibodies against Candida tropicalis mannans antigen detection by enzyme immunoassay and immunofluorescence. J Clin Microbiol 1986; 24: special stains and h & E