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Stains & CD markers
GMS

Author: Fang Zhou, M.D.

Deputy Editor-in-Chief: Patricia Tsang, M.D., M.B.A.

Last author update: 7 May 2021

Last staff update: 5 February 2024

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PubMed Search: Gomori methenamine silver "last 5 years"[dp]

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Table of Contents

Cite this page: Zhou F. GMS. PathologyOutlines.com website. https://www.pathologyoutlines.com/topic/stainsgms.html. Accessed May 2nd, 2024.

Definition / general

Grocott-Gomori methenamine silver (GMS) is a special stain to detect fungi
Also positive in some nonfungal organisms and nonorganisms

Essential features

Outlines fungal organisms by staining polysaccharides in cell walls
Fungal morphology on GMS usually not specific enough to allow definitive species identification
Stains some nonfungal organisms: bacteria, mycobacteria, Strongyloides, viral inclusions
Artifacts, mimickers and other pigments are common confounders to accurate interpretation

Terminology

Other names for GMS:
- Grocott stain
- Grocott methenamine (hexamine) silver stain
Disambiguation from other similar terms:
- Other silver stains (e.g. Warthin-Starry, Jones, Steiner)
- Giemsa stain

Pathophysiology

Aldehyde groups form on polysaccharides (1,2-glycols) via oxidation by chromic acid (Suvarna: Bancroft's Theory and Practice of Histological Techniques, 8th Edition, 2019, IHC World: A Common Mistake When Staining for Fungi [Accessed 31 March 2021])
- Aldehyde groups are further oxidized over time
- Fungal cell walls (high polysaccharide density) retain more aldehyde groups
- Background structures, such as basement membranes, collagen and reticulin (lower polysaccharide density) retain fewer aldehyde groups
- Aldehyde groups reduce silver nitrate (or Schiff molecules) to create a metallic silver precipitate which appears gray to black
Weaker oxidizers like periodic acid (which is used in PAS and Jones stains) have more staining of background structures

Clinical features

GMS stain cannot provide definitive organism identification
Identification is preferably by other methods:
- Culture (which can also gather susceptibility data)
- Alternative testing of nontissue samples (e.g. antibody detection, antigen detection, galactomannan, β-D-glucan, PCR, blood culture, etc.)
- Molecular testing
  - Few clinically validated tests are available for testing of formalin fixed, paraffin embedded (FFPE) blocks
  - Many false negatives due to formalin crosslinking
- In a few instances, additional special stains on formalin fixed, paraffin embedded (FFPE) blocks
Reference: Clin Microbiol Rev 2011;24:247

Interpretation

Sharply outlines fungal cell walls
Gray to black with peripheral outlines
Organisms may be very few and pale
Artifacts are common
- Irregular sizes and shapes
- No peripheral outline
- Not associated with tissue reaction
- Out of tissue plane
Counterstain: light green
- Can also use light H&E (e.g. when a case sent for consult contains only one unstained slide)
Signs of infection: inflammation with or without granulomas, necrosis, hemorrhage, angioinvasion
Otherwise, it may be colonization rather than true infection; clinical correlation is required
In comment, describe the fungal elements seen on GMS
- Hyphae: Septated or nonseptated? Branching or nonbranching? Acute or right angle branching?
- Yeasts: Budding or nonbudding? Broad based or narrow based budding?
- Presence of spherules or endospores
If a fungal identity in the report is requested by clinician, then state the fungi most frequently associated with this morphology and that morphologic mimickers exist; include a statement about the importance of clinical correlation
Reference: Clin Microbiol Rev 2011;24:247

Uses by pathologists

GMS is ordered when fungi are suspected on H&E or cytology preparations
- Granulomatous reaction is most common
- Wide range of other reactions: exudative, necrotic or little cellular response (immunocompromised)
- On H&E and cytologic preparations, fungi can be stained at varying intensities, appear refractile (capsule) or may not be visible
- More than one organism may be identified in the same lesion
Advantages
- Relatively quick and cost effective
- GMS positive fungus in tissue helps confirm a positive culture result for environmentally ubiquitous fungi (versus contaminant)
- Culture may be false negative
- Pneumocystis cannot be grown in culture (mBio 2018;9:e00939)
- Antibody testing can be negative in immunodeficient patients
- Antibody and antigen tests may crossreact between different organisms
Disadvantages
- Low sensitivity
- Cannot definitively identify fungal species
  - Many morphologic mimics (Clin Microbiol Rev 2011;24:247)
  - Morphology may be changed by treatment, biopsy procedure or variant forms
- Cannot provide antifungal susceptibility data

Microscopic (histologic) images

Contributed by Fang Zhou, M.D.

Positive staining: normal

Keratin debris

Cytoplasmic mucin, peribronchial glands

Cytoplasmic mucin, sinonasal mucosa

Thin extracellular mucin

Thick extracellular mucin

Neutrophil cytoplasm

Foreign bodies near fungus

Artifacts out of plane

Polarizable foreign fibers

Red blood cells overstaining

Positive staining: fungal organisms

Fungal hyphae

Fungal yeasts

Fungal pseudohyphae and yeasts

Probable Cryptococcus spp.

Positive staining: nonfungal organisms

Gram positive coccal bacteria

Filamentous bacteria

Mycobacteria

Other confounding pigments

Calcium phosphate deposits

Melanin pigment

Anthracotic pigment

Hemosiderin pigment

Contributed by Andrey Bychkov, M.D., Ph.D. and Jijgee Munkhdelger, M.D., Ph.D.

Candida esophagitis

Esophagitis

Virtual slides

Images hosted on other servers:

Invasive aspergillosis, sinonasal

Deep fungal infection, skin

Fungal dermatitis

Positive staining - normal

Intracellular and extracellular mucin (J Am Soc Cytopathol 2017;6:223, Am J Clin Pathol 1946;10:177)
Glycogen: can predigest with diastase but will also decrease sensitivity for fungi (IHC World: A Common Mistake When Staining for Fungi [Accessed 31 March 2021], Am J Clin Pathol 1946;10:177)
Neutrophil cytoplasm (J Cytol 2019;36:184)
Elastin: from overincubation (Suvarna: Bancroft's Theory and Practice of Histological Techniques, 8th Edition, 2019)
Other pigments (Clin Microbiol Rev 2011;24:247)
- Anthracotic pigment: coarse granules, completely black, no cell wall outlines, remains black on GMS
- Melanin: fine to coarse brown granules on H&E, turns black on GMS
- Hamazaki-Wesenberg bodies: yellow-brown oval structures seen in sarcoidosis on H&E, turns black on GMS
Red blood cells (aberrant staining)

Positive staining - disease

Fungal yeasts (diameter 2 - 15 microns) (Clin Microbiol Rev 2011;24:247)
- Blastomyces dermatitidis
- Cryptococcus spp.
- Histoplasma capsulatum
- Candida glabrata: yeasts only (other Candida spp. show mixed yeasts and pseudohyphae)
- Pneumocystis jirovecii: will not grow in culture
- Penicillium marneffei
- Paracoccidioides brasiliensis
- Sporothrix schenckii
- Emmonsia crescens
- Lacazia loboi
- Other emerging opportunistic fungi, etc.
Fungal hyphae (diameter 2 - 6 microns) (Clin Microbiol Rev 2011;24:247)
- Often ubiquitous in the environment (especially soil) and can contaminate cultures, so seeing hyphae in infected tissue is helpful to confirm culture results (Clin Microbiol Rev 2011;24:247)
- Aspergillus spp.
- Mucorales order: stains poorly with GMS; add PAS stain
  - Mucor spp. and Rhizopus spp. (mucormycosis / zygomycosis)
  - Entomophthorales spp.
- Dematiaceous (melanin+) fungi
  - Madurella spp., Fonsecaea spp., Cladophialophora spp., Exophiala spp., Curvularia spp., Bipolaris spp., etc.
- Other hyphae seen in tissue infections: Trichosporon spp., Fusarium spp., Scedosporium spp., Trichoderma spp., Paecilomyces spp., Pseudoallescheria spp., Scopulariopsis spp., Acremonium spp., Schizophyllum spp., Phaeoacremonium spp., Trichophyton spp., Phialophora verrucosa, Bipolaris spicifera, Curvularia lunata, etc. (other emerging opportunistic fungi)
Mixture of fungal yeasts and pseudohyphae:
- Candida spp. (except C. glabrata) (Clin Microbiol Rev 2011;24:247)
Mixture of fungal spherules and endospores:
- Coccidioides spp.
  - Spherules (10 - 100 microns) and endospores (2 - 5 microns)
  - Endospores morphologically resemble yeasts
  - Spherules are not always found
- Coccidioides immitis rarely infects tissues as spherules and hyphae (Clin Invest Infect 1994;105:P412, Clin Infect Dis 2000;30:349, Am J Trop Med Hyg 2019;100:772)
Nonfungal organisms (J Am Soc Cytopathol 2017;6:223, Diagn Cytopathol 2017;45:1105, Suvarna: Bancroft's Theory and Practice of Histological Techniques, 8th Edition, 2019, Clin Microbiol Rev 2011;24:247)
- Must tell them apart from fungi (use size, shape)
- May be helpful in challenging or sparse cases to prompt the ordering of additional validated confirmatory stains or clinical tests for the nonfungal organisms
  - In absence of other confirmatory results, interpret the GMS with caution (contaminants, artifacts, mimickers)
- Mycobacterium
  - Fatty capsule may be lost due to antimycobacterial therapy and tissue processing, causing acid fast stains to fail or stain fewer bacilli
  - Sometimes, GMS can still stain residual cell wall carbohydrates
- Bacteria (Gram stain may be lost due to necrosis or antibiotics)
  - Streptococcus spp.
  - Staphylococcus spp.
  - Filamentous: Nocardia and Actinomyces spp.
  - Bacillus cereus
  - Bartonella henselae
  - Bartonella quintana
- Strongyloides stercoralis
- Schistosoma eggs
- Cytomegalovirus inclusions
- Toxoplasma

Negative staining

Organisms mimicking fungi that are GMS negative (Clin Microbiol Rev 2011;24:247)
- Rhinosporidium seeberi
- Trypanosoma cruzi
- Leishmania spp.
Nonorganisms (may resemble fungi on H&E but do not contain carbohydrates and are negative for GMS)
- Calcium phosphate and calcium carbonate deposits: associated with necrosis; mimic yeasts (Dako: Special Stains and H&E [Accessed 1 April 2021])
- Gamna-Gandy bodies: associated with blood - iron and calcium salts; mimic hyphae (Diagn Cytopathol 2020;48:670)
- Hemosiderin: mimics yeasts
- Frothy fibrin or edema: mimic yeasts
- Crystals and precipitates: mimic yeasts or hyphae

Sample pathology report

Sinonasal mucosa, biopsy:
- Invasive fungal sinusitis (see comment)
- Comment: GMS stain on block A1 shows broad, nonseptate fungal hyphae with right angle branching, associated with tissue necrosis and angioinvasion, consistent with invasive fungal sinusitis. Dr. _ was notified at 10:00am on 4/19/2021 with repeat back verification.

Board review style question #1

GMS stain performed on an esophageal biopsy from a patient with AIDS shows a mixture of yeasts and pseudohyphae, suggestive of Candida species. Which species can be ruled out based on this morphology?

Candida albicans
Candida dubliniensis
Candida glabrata
Candida parapsilosis
Candida tropicalis

Board review style answer #1

C. Candida glabrata, formerly known as Torulopsis glabrata, is the only Candida species that does not form pseudohyphae in tissue. It was formerly considered a saprophytic organism that is commonly found in normal human flora but it has emerged as an opportunistic organism with both innate and acquired antifungal resistance (Microorganisms 2019;7:39, J Clin Microbiol 2004;42:4419).

Comment Here

Reference: GMS

Board review style question #2

A biopsy from a solid lung nodule shows granulomatous inflammation and fungal hyphae are seen on GMS stain. Which of the following is the most likely causative organism?

Blastomyces dermatitidis
Cryptococcus spp.
Histoplasma capsulatum
Pneumocystis jirovecii
Scedosporium spp.

Board review style answer #2

E. Among the listed organisms, only Scedosporium spp. (though uncommon) are usually encountered as hyphae in infected tissue specimens (Clin Microbiol Rev 2008;21:157). It is an opportunistic fungus with intrinsic antifungal resistance (Med Mycol 2018;56:102). The other organisms in the answer choices are more commonly encountered dimorphic fungi that are usually seen as yeast forms in infected tissues.

Comment Here

Reference: GMS

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