General principles of mycology

Specimen collection and processing

Can refer to the HRLMP Laboratory Test Information Guide.
Skin, hair, nail for dermatophytes
- Skin: clean with 70% alcohol. Scrape off the surface, the collect a subsurface sample from the inflammatory edge of the lesion, using a scalpel or a dermal curette.
- Nail: clean an unpolished nail with 70% alcohol. Scrape the surface clear, then collect a sample of lower nail table/plate at the edge of lesion with a small curette or scalpel.
- Hair: 10-12 hair roots with surrounding crust, with sterile forceps, placed in a clean envelope or sterile cup.
- Should be submitted without added moisture
Tissue: the preferred sample for fungal culture. Should be placed in a small amount of preservative-free saline to keep it moist
Processing:
- Often directly inoculated onto culture media.
- Can also mince (with scalpel) or homogenize (grind) to improve yield.
  - Mince better for fungal to preserve structure.

Fungal media

Review CLSI M54-A.
Sabouraud with and without antibacterials: originally made for dermatophyte growth, also useful for yeasts. Not good for dimorphics.
- Chloramphenicol and gentamicin added to inhibit growth of bacteria.
- Cycloheximide added to inhibit growth of rapidly-growing contaminating molds.
  - However, will inhibit Mucorales, so always need media with and without cycloheximide.
- Inhibitory antimicrobials not needed if collected from a sterile site.
BHI (blood-heart infusion) with blood: enriched medium. Good for yeasts, including Cryptococcus, and dimorphic fungi. Non-selective medium. Used for saprophytic and dimorphic fungi (cycloheximide inhibits saprophytes).
Sabouraud’s BHI (SABHI): also exists, good for fastidious fungi.
Littman: selective medium for fungi. Crystal violet and streptomycin inhibit bacterial growth.
Inhibitory Mould Agar (IMA): used to grow dimorphic fungi. Saprophytic and dermatophytic fungi will not grow. Can be used instead of SAB.
Potato flake agar: used for fastidious and slow-growing strains.
Cycloheximide in primary media: prevents growth of rapidly-growing molds that are frequently contaminants. However, it does inhibit some pathogenic fungi including Mucorales, Cryptococcus, Candida krusei, other Candida spp., Trichosporon spp., P. boydii, and Aspergillus spp.

Selecting appropriate media

Bronchoscopy or lung tissue: SAB-CG, Littman, and BHIA-CCG w 10% SB; heavy inoculum; for BAL >2mL, centrifuge down an inoculate 0.5 ml/plate.
- Can add Gram stain and CAN-BA for Nocardia.
- Can add PJP stuff for PJP.
- Target organisms include:
  - Hyaline molds: Aspergillus, Fusarium, Penicillium, Scedosporium
  - Mucorales
  - Yeasts: Cryptococcus
  - Dimorphics: Histo/Blasto/Crypto
Vaginal swab: SAB-CG and Littman, to help isolate Candida.
- Chromogenic agar may be useful e.g. BioRad CandiSelect-4 (C. albicans is purple, others are green)
Inducing sporulation: Potato dextrose agar (PDA)
Suspected histoplasmosis: Incubate for 28 days at 30ºC for as long as 6 weeks.

Classification of fungi

Working classification

Yeast
Filamentous fungi
- Hyalohyphomycosis (hyaline molds)
- Mucormycosis
- Dematiaceous / phaeohyphomycosis (pigmented hyphae)
- Endemic mycoses / dimorphic fungi
- Dermatophytes (also hyaline, but separated for simplicity)
Pneumocystis
Unusual fungal and fungal-like

Four Phyla

Zygomycota/Glomeromycota: Mucormycotina and Entomycophthomycotina? e.g. Mucorales
Ascomycota: e.g. Candida, Fusarium, Sporothrix, Aspergillus, Penicillium, Coccidioides, Histoplasma
Basidiomycota: e.g. Cryptococcus
Deuteromycota
Other: Blastomyces, “black yeasts” (informal)

Clinical Classification

Superficial/cutaneous mycoses, e.g. dermatophytes, tinea, etc.
Subcutaneous mycoses, e.g. mycetoma, sporotrichosis
Systemic mycoses, e.g. Aspergillus, dimorphics, Candida
Opportunistic mycoses, e.g. same as systemic, also Cryptococcus, mucormycosis

Automated blood culture systems

BacT/Alert is an automated blood culture system that uses enriched trypticase soy broth, automatically monitored for growth with a CO2 sensor.
Isolator lysis-centrifugation tube has components to lyse RBCs and WBCs, inhibit the immune cell actions; tubes are lysed then centrifuged and the sediment plated onto agar media. Isolator can release any intracellular fungi.
Per Am J Clin Pathol 1995;103(5):660-662, the Isolator system is much more sensitive for fungi in general, though Candida spp. are fairly well-detected by BactT/Alert.

System	CO₂	Temp	Overall Yields	Better For	Speed
BacTAlert	5%	36ºC	Not significantly different	Aerobic and facultatively anaerobic Gram-negative bacilli, Candida, Cryptococcus, and Fusarium	Faster if organism detected in both blood cultures
Isolator System (lysis centrifugation)	5%	37ºC	Not significantly different	Staphylococcus spp., yeast (C. parapsilosis, C. lusitaniae, Rhodotorula, and Cladosporium), dimorphic fungi (Histoplasma), and M. furfur	Faster identification of organisms on solid media

Histology of fungi

Review Am J Clin Pathol 2009;131:364-375: histology alone cannot reliably identify fungi beyond routine descriptions like “hyaline filamentous fungus”
Approach to histology:
- Yeast vs molds / pseudohyphae vs hyphae
- Size or width
- Septations
- Branching

Stains

Regular H&E (hematoxylin and eosin): though not reliable for fungi. Can stain some fungal cells purple but is mostly used to identify the inflammatory response in the tissue
Periodic acid-Schiff (PAS): complex carbohydrates in fungal cell wall will stain red/purple/pink. Diastase may be used to digest glycogen in tissue (which also stains with PAS).
Gomori methenamine silver (GMS): a silver precipitation stain that is sensitive for fungi (possibly better than PAS). Fungi are stained black.
Mucicarmine: stains mucins as well as Cryptococcus polysaccharide capsule bright red.
Fontana-Masson: detects melanin and related pigments in fungi in dematiaceous fungi and Crypto. neoformans/gattii.

Invasive fungi

Invasive Aspergillosis: septate hyphal filaments (5-10µm thick) with dichotomous branching at 45º. May see fruiting bodies in lung cavities. Pulmonary lesions appear as nectrotizing pneumonia with sharply delineated gray foci and hemorrhagic borders (called target lesions).
Differentiating IA from other molds: Aspergillus hyphae cannot be distinguished from Pseudallescheria boydii and Fusarium spp. by histology alone; not reliably distinguished from Zygomycetes, either. Fruiting bodies may be more helpful, if seen. Culture is needed.
In tissue samples it can be very difficult to tell them apart Rapid (<3 hours) ribosomal RNA in situ hybridization (ISH) protocol using dual fluorogenic-labeled oligonucleotide probes was developed. These probes are composed of a mixture of DNA and locked nucleic acids
- Aspergillosis: Stains positive with Masson-Fontana
- Hyaline Moulds: i.e. Penicillium, Fusarium, Scopulariopsis, Pseudallescheria, Scedosporium, Acremonium, Paecilomyces, and Trichoderma species.
  - When differentiating between this and Aspergillus, other than Scedosporium, Aspergillus will stain positive with Masson-Fontana
- Candida: on gram stain, yeast cells and pseudohyphae will appear gram-positive on clinical specimens
- Zygomycetes: Mucor velutinosus does not stain with GMS
- Actinomyces: stains gram-positive with the Brown-Brenn Stain
- Nocardia: stains gram-positive with the Brown-Brenn Stain
- PJP, H.capsulatum: stains with GMS vs Leishmania which does not stain with GMS
- Cryptococcus: Stains burgundy red with Mayer’s mucicarmine stain
- Blastomyces dermatitidis: can faintly stain with Mayer’s mucicarmine

Direct microscopy of fungi

KOH-Calcofluor: used for the direct examination of fungi. KOH clears tissue cells and helps the calcofluor, which binds the chitin and cellulose in fungal cell walls. Under UV light, the fungi will fluoresce.
- No patient preparation required prior to collection of the sample
- Step 1: Collect the sample i.e. skin, nails, hair
- Step 2: The sample is then placed directly onto a microscope slide
- Step 3: You then cover the sample with 10% or 20% KOH
- Step 4: Wait until the sample is completely dissolved (5-15 minutes)
- Step 5: Addition of Lactophenol cotton blue stain to better visualize the fungus
- Step 6: Add calcofluor-white stain to the slide to cause the fungi to become fluorescent
- Step 7: Identify the fungus via microscope
India ink: used to identify capsules. The ink floods the slide except where the capsule is present, creating a halo around encapsulated organisms such as Cryptococcus.
- False positives with WBCs (halo diminishes with time) or contamination of the India ink itself.
Gram stain can still demonstrate yeasts.

Textbook References

Larone Medically Important Fungi 5^th edition
St. Germain & Summerbell’s Identifying Fungi
Anaissie Clinical Mycology 2^nd edition
ASM Bench Manual for Mycology
ESCMID and ECMM Clinical Guidelines Series, in Clin Microbiol Infect 2014;20(Suppl. 3)
Lancet Infectious Diseases Series on Fungal Infections, in issues 2017;17(11) and 2017;17(12)
IDSA Guidelines