Fungal Infections

Histopathology

Demonstration of fungi in tissue is confirmatory of fungal infection. So the 2008 EORTC/MSG definitions of invasive fungal infection[i] carry these statements for proven infections;

  •   Moulds: “Histopathologic, cytopathologic, or direct microscopic examination of a specimen obtained by needle aspiration or biopsy in which hyphae or melanized yeast-like forms are seen accompanied by evidence of associated tissue damage.”
  •   Yeasts: “Histopathologic, cytopathologic, or direct microscopic examination of a specimen obtained by needle aspiration or biopsy from a normally sterile site (other than mucous membranes) showing yeast cells—for example, Cryptococcus species indicated by encapsulated budding yeasts or Candida species showing pseudohyphae or true hyphae.”

Parallel processing to minimize delay

The BSMM Standards of Care (2003)[ii] set out 2 standards for histopathology. The first is: “All tissues from immunocompromised (including corticosteroid-treated) patients and those with suspected fungal infection should be stained with fungal stains such as periodic acid-Schiff, silver, or fluorescent stains, in parallel with regular stains. Biopsy and surgical resection of abnormal tissue are frequently definitive features in the diagnosis of invasive fungal infections. Speed is critically important in achieving an early diagnosis, and the practice of assessing haematoxylin and eosin (H&E) stains of tissues before deciding whether to use specialised stains for fungi frequently introduces fatal delays for patients. Clear indication on all pathology request cards that the patient is immunocompromised is critically important.” 

Standard stains

Standard histological stains for fungi are Grocott (methanmine) silver (GMS) stain or Periodic acid-Schiff (PAS). The GMS stain is more sensitive than the PAS stain, but has a signal to noise issue in that it stains inflammatory cells (lysosomes) and tissue reticulin, in addition to fungi. PAS staining has the slight advantage that the morphology of the tissue adjacent to the fungi can be better visualized, but this can be addressed using a GMS stain and H&E counterstain. Inclusion of good control sections is mandatory as some fungi such as Mucorales may require longer staining times and other fungi can be ‘over-stained’.

On H&E, all fungi show pink cytoplasm, blue nuclei and no colouration of the wall. Tissue Gram stains can stain Candida spp. blue, consistent with a Gram positive organism. With an acid-fast stain for Mycobacteria, Blastomyces and Histoplasma can be appear red and be acid-fast staining.

Suspecting a fungal infection histologically

Sometimes a fungal infection is not suspected clinically, and the interpreting pathologist has to suspect it. Sometimes fungal hyphae or other structures such as spherules are directly visible, but scanty hyphae and small yeasts are easily missed. The host response to fungi is very varied partly depending on whether the patient is immunocompetent or immunocompromised. No host cell reaction at all may be seen in imunocompromised patients especially with pulmonary Pneumocystis and disseminated Cryptococcus infections. Clues to the diagnosis of a fungal infection are:

  • Mixed granulomatous reaction, seen with both yeast and filamentous fungal infection;
  • Granulomas with caseation necrosis, mimicking mycobacterial infections seen with Histoplasma and Coccidioides infections;
  • Vascular invasion and secondary thrombosis with infarction and haemorrhage seen especially in neutropenic patients with Aspergillus or Mucorales infection;
  • Epithelial hyperplasia of skin with mixed inflammation and abscesses within the epidermis, seen with cutaneous fungal infection.

Reporting results

The first key requirement of histopathological reporting is confidence that a fungal infection is or is not present in the tissue sample. Compared with culture or other diagnostic testing, the sensitivity of histology is at best 80% (and often as low as 20%, especially if specialised GMS or PAS stains are not done). However, negative reports are valuable, despite the less than perfect sensitivity, as some cultures and antigen tests are falsely positive. Diagnosing a fungal infection, as opposed to malignancy, tuberculosis, leishmaniasis or auto-immune disorder (to name some differential diagnoses) has major implications for each patient.

Histopathologists should report both the fungal morphology and the tissue reaction to it; ie what they see in tissue2. The histological reaction often determines the precise diagnosis, such as granulomatous fungal rhinosinusitis. Histopathologists should describe whether the fungal morphology is yeast or hyphal (or both as in dimorphic infection) and the yeast or hyphal diameter. They should note the type of budding of the yeast - narrow or broad-based, or fission. For hyphae, they should note whether the hyphal form is regularly septated or not, are the hyphae the usual width of Aspergillus hyphae (3-12um) or wider, branching angle or have a dilated, bizarre shape, more consistent with Mucorales. They should also note if any surrounding brown pigmentation is present (dematicaeous) or are unpigmented colourless/pale blue on H&E stain (hyaline). An excellent recent resource was published by Guarner and Brandt in 2011[iii].

Some fungi are highly distinctive and what is seen is sufficient to make a definitive aetiological (fungal) diagnosis. See Table 1. Others are less so, in particular filamentous fungi. The number of species involved is large, although Aspergillus fumigatus remains the most common mould infection worldwide. Candida spp. with the common exception of C. glabrata, are dimorphic, so that both yeast cells and hyphae are visualised together. In heavily immunocompromised patients, mixed infections are now seen in up to 20% of patients, further complicating interpretation of positive findings. A few key distinctions are important for immediate treatment decisions, notably:

  •   Distinguishing hyphae characteristic of Mucorales, from other hyphae, as all Mucorales are resistant to fluconazole, itraconazole, voriconazole and echinocandin antifungals
  •   Identifying Pneumocystis jirovecii, which is generally azole, amphotericin B and echinocandin resistant.
  •   Distinguishing the small yeasts without hyphae characteristic of Candida glabrata, which is fluconazole resistant.
  •   Distinguishing Cryptococcus spp. from Candida spp, as Cryptococcus spp. are echinocandin resistant.

Table 1

Distinctive fungi seen on histopathology, sufficient to make a definitive aetiological diagnosis

Appearance

Tissue reaction

Fungal infection

Narrow-based budding yeasts (4–10µm) with a thick capsule

None or granulomatous reaction, occasionally fibrosis.

Cryptococcus spp.

Broad-based budding yeasts (10–15µm)

Mixed suppurative and granulomatous inflammation typical.

Blastomyces dermatitidis

Small yeasts (2–4µm) clustered inside macrophages with narrow-based budding

Several appearances depending on chronicity of infection; vascular necrosis and granulomatous inflammation.

Histoplasma capsulatum

Small oval yeasts (2–5µm) with a transverse septum, indicating division by fission.

Mixed suppurative and granulomatous inflammation or necrosis with little reaction.

Penicillium marneffei

 

Round, oval, or cigar-shaped yeasts (2-6+µm) with narrow-based or tube-like budding.

 

Mixed suppurative (including eosinophils) and granulomatous inflammation, Hoeppli-Splendore phenomenon frequent.

Sporothrix schenckii

 

Spherules (10-100µm) with multiple endospores (2-5µm), (often confused with yeasts).

Mixed suppurative (including eosinophils) and granulomatous inflammation with a rim of lymphocytes, Hoeppli-Splendore phenomenon likely.

Coccidioides immitis/posadasii

Spherical large yeasts (4-60 µm) surrounded by multiple buds [ship’s wheel] with an optically clear space between the fungus and surrounding tissue.

Yeasts are usually found inside multinucleated giant cells.  Mixed suppurative and granulomatous inflammation surrounded by fibrosis

Paracoccidioides brasiliensis

Special stains

Several special stains are used to exemplify certain characteristic features of fungi. These are listed in Table 2.

Table 2 - Specialised stains for fungi.

Stain

Utility

Interpretation

Mucicarmine

Showing the polysaccharide capsule of Cryptococcus spp.

Bright pink surrounding a medium sized yeast, confirms that capsular polysaccharide is present.

Alcian blue

Showing the polysaccharide capsule of Cryptococcus spp.

Stains the capsule blue.

Fontana-Masson

Stains cell wall and extracellular melanin

Useful to demonstrate that the fungus is a melanin producer, as in dematiaceous fungi and Cryptococcus. Caution should be exercised when interpreting Fontana-Masson staining, since many Aspergillus spp., some Mucorales genera, and Trichosporon can also show positive staining.

Cresyl fast violet

Stains bacteria and fungi

Most fungi appear blue or purple, Mucorales may appear red.

Immunohistochemistry for fungal identification

Immunohistochemistry (IHC) or immunocytochemistry is a method for localizing specific antigens in tissues or cells based on antigen-antibodies recognition. Specificity should derive from binding of an antibody with its counterpart antigen at a light microscopic level. The use of an enzymatic label antibody such as horseradish peroxidase allows visualization of the labeled antibody using conventional light microscopy in the presence of a suitable chromogenic substrate system. IHC is now applied to routinely formalin-fixed paraffin-embedded tissues sections. Depending on the colorimetric developer, fungi stain dark brown or red, usually with a counterstain of hematoxylin. Immunohistochemical methods have the potential advantage of providing a rapid and specific identification of several fungi, allowing pathologists to identify unusual filamentous and yeast-like infections and accurately distinguish them from confounding artefacts.

IHC for Candida, Aspergillus, Blastomyces, Cryptococcus, Histoplasma, Coccidioides, Pneumocystis, Sporothrix, Paracoccidioides, Penicillium and Mucorales have been developed and some are available commercially. For the identification of Aspergillus species, polyclonal and monoclonal antibodies have been produced but most of these antibodies cross-react with other fungi. Cross-reactivity of the antibodies is a common problem limiting their use. None are CE-marked or FDA-approved.

In situ hybridization for fungal identification

In situ hybridization (ISH) uses molecular probes for different fungi to contribute to identification. Most probes are ribosomal to amplify the tissue signal. Depending on the colorimetric developer, fungi stain dark brown or red. Probes published in the literature include those for Candida, Aspergillus, Blastomyces, Cryptococcus, Histoplasma, Coccidioides, Pneumocystis, Sporothrix, Mucorales, Fusarium and Pseudallescheria.

The accuracy of ISH appears to be high but sensitivity on formalin-fixed tissue is reduced.

Direct sequencing from tissue for fungal identification

Fungal DNA can be amplified directly from tissue (preferably fresh), using broadrange PCR assays targeting ribosomal RNA genes, especially the ITS 1 or ITS 2 regions, or 18S or 28S. DNA extraction and PCR methods are published allowing amplification of DNA from many diverse fungi in the presence of human DNA. The PCR product can then be sequenced and sequences compared with the publically available databases. This allows direct identification, although DNA extraction and amplification fails in up to 50% of cases from formalin-fixed tissue.

References:
[i] De Pauw B, Walsh TJ, Donnelly JP, Stevens DA, Edwards JE, Kauffman C, Calandra T, Pappas P, Bille J, Denning DW, Dismukes WE, Herbrecht R, Hope WW, Kibbler CC, Kullberg BJ, Lortholary O, Maertens J, Marr K, Maschmeyer G, Patterson TE, Perfect J, Restrepo J, Ruhnke M, Brahms B, Sobel J, Sorrell TC, Viscoli C, Wingard JR, Zaoutis T, Bennett JE. Defining invasive fungal diseases for clinical research: revised definitions of the EORTC/MSG Consensus group. Clin Infect Dis 2008:46:1813-21.

[ii] Denning DW, Kibbler C, Barnes R. British Society for Medical Mycology proposed standards of care for invasive fungal infections. Lancet Infect Dis 2003;3:230-40.

[iii] Guarner J, Brandt ME. Histopathologic diagnosis of fungal infections in the 21st century. Clin Microbiol Rev 2011;24:247-80.
 

Guarner J. Incorporating Pathology in the Practice of Infectious Disease: Myths and Reality. Clin Infect Dis 2014; pii: ciu469.
 

Resources

Jensen HE & Chandler FW. Histopathological diagnosis of mycotic diseases: In: Topley and Wilson's Microbiology and Microbial Infections. 10th Edition.  John Wiley & Sons, Ltd, 2010.
Chandler FW & Watts JC. Pathologic Diagnosis of Fungal Infections. ASCP Press, Chicago, 1987

Images

Comparison of GMS and PAS stains. Patient with disseminated Trichosporon spp. infection. Both x60. In the GMS image, substantial background staining of elastin is seen, with more prominent yeasts superimposed. In contrast, the PAS stain shows the tissue morphology, with bright pink yeasts also visible.

GMS stain

PAS stain. An example of Aspergillus fumigatus 


(PAS-stained) in a patient with chronic granulomatous disease showing a 45 degree branching hypha within a giant cell. Rather bulbous hyphal ends are also seem, which is sometimes found in Aspergillus spp. infections, histologically. (x800)

PAS-stainAn example of Aspergillus fumigatus in a patient with chronic granulomatous disease showing a 45 degree branching hypha within a giant cell. Rather bulbous hyphal ends are also seem, which is sometimes found in Aspergillus spp. infections, histologically. x800

Gram stain. Candida albicans in a section of lung, confirming an invasive Candida pneumonia. Both yeasts and hyphae are visible. Candida is staining Gram positive.

H&E stain. An example of dematiacous fungi causing chromoblastomycosis. Strings of fungal cells, of 2 different sizes, with brown melanin on the fungal cell surface. (x600)

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