Special Stains Use in Fungal Infections - Dako

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192 | Connection 2010 Figure 9. Hematoxylin and Eosin staining of Histoplasma. Figure 10. AFB staining of Histoplasma.
“ 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. ” 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 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). � Connection 2010 | 193
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Special Stains Use in Fungal Infections - Dako

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