Lab diagnosis for general mycosis1

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Laboratory Diagnosis of General Mycosis

The laboratory diagnosis of fungal infections (mycoses) relies on a combination of direct microscopy, culture, histopathology, serology, and molecular methods. Below is a systematic overview.

1. Specimen Collection

The type of specimen depends on the clinical category of mycosis:

Mycosis Type	Typical Specimens
Superficial	Skin scrapings, hair clippings, nail clippings
Cutaneous	Skin scrapings (deeper epidermis), hair, nails
Subcutaneous	Biopsy, pus/drainage from sinus tracts
Systemic/Opportunistic	Blood, BAL fluid, CSF, bone marrow, urine, biopsies

Key rule: Specimens must be transported promptly and kept at room temperature (NOT refrigerated for molds).

2. Direct Microscopy

KOH (Potassium Hydroxide) Preparation

The most widely used initial test
10-20% KOH dissolves keratin and host cells, leaving fungal elements visible
Used for skin scrapings, nail clippings, hair samples
Reveals: hyphae, pseudohyphae, yeast cells, spores, arthroconidia
Malassezia shows the classic "spaghetti and meatballs" pattern (short hyphae + spherical yeast cells)
KOH + calcofluor white (fluorescent brightener) increases sensitivity - binds chitin in fungal cell walls and fluoresces under UV

Wet Mount / India Ink (Nigrosin) Preparation

India ink highlights the capsule of Cryptococcus neoformans in CSF - the capsule appears as a clear halo against the dark background
Sensitivity ~50% in HIV-positive patients; superseded by the cryptococcal antigen test but still useful

Gram Stain

Candida spp. appear as gram-positive budding yeasts and pseudohyphae
Not useful for most molds

Giemsa / Wright Stain

Useful for demonstrating Histoplasma capsulatum inside macrophages in bone marrow or peripheral blood smears (small intracellular yeasts)

3. Culture

Culture is the gold standard for definitive diagnosis.

Media

Sabouraud Dextrose Agar (SDA): Standard medium with low pH (5.6) and glucose - inhibits most bacteria but supports fungal growth. Incubated at 25-30°C
SDA + antibiotics (chloramphenicol/cycloheximide): Inhibits bacteria and saprophytic fungi; used for dermatophytes
Brain Heart Infusion (BHI) agar: Enriched medium for fastidious fungi and dimorphic fungi at 37°C (yeast phase)
Mycosel/Mycobiotic agar: Contains cycloheximide to suppress contaminants
CHROMagar Candida: Chromogenic medium that differentiates Candida species by color (C. albicans = green, C. tropicalis = blue, C. krusei = pink)

Incubation

Cultures must be held for 4-6 weeks before being reported negative (many pathogenic fungi grow slowly)
Dermatophytes: 1-2 weeks at 25°C
Dimorphic fungi (Histoplasma, Blastomyces, Coccidioides): 4-6 weeks, BSL-3 precautions required

Colonial Identification

Colonies are identified by:

Macroscopic features: Color (surface and reverse), texture (powdery, velvety, cottony, waxy), growth rate, topography
Microscopic features (Lactophenol Cotton Blue mount): Morphology of hyphae (septate vs. aseptate), type of conidia (micro vs. macroconidia), conidiophores, sporangia, special structures (chlamydospores, cleistothecia)

4. Histopathology

Tissue biopsy with special stains is essential for invasive/systemic mycoses.

Stain	Uses
Hematoxylin & Eosin (H&E)	General tissue morphology; fungi poorly visible
Periodic Acid-Schiff (PAS)	Stains fungal cell walls magenta/red; excellent for yeasts and hyphae
Gomori Methenamine Silver (GMS / Grocott)	Stains fungi black against green background; most sensitive for demonstrating fungi in tissue
Mucicarmine	Stains Cryptococcus capsule red
Fontana-Masson	Detects melanin in dematiaceous fungi (chromoblastomycosis)

5. Serology and Antigen Detection

These tests are especially important for systemic/disseminated mycoses, where culture can be slow or insensitive.

Test	Pathogen	Notes
Cryptococcal Antigen (CrAg) - Latex Agglutination or LFA	Cryptococcus neoformans/gattii	Highly sensitive/specific; serum or CSF; lateral flow assay allows point-of-care use
Galactomannan (GM) ELISA	Aspergillus spp.	Beta-1,5-galactofuranan antigen; serum or BAL; useful for invasive aspergillosis in neutropenic patients
(1→3)-β-D-Glucan Assay	Pan-fungal (NOT Cryptococcus, NOT Mucorales)	Detects cell wall component of most fungi; useful screening test
Histoplasma Urine Antigen	Histoplasma capsulatum	Most sensitive for disseminated and acute pulmonary histoplasmosis
Blastomyces Urine Antigen	Blastomyces dermatitidis	Cross-reactivity with Histoplasma
Coccidioides Complement Fixation / ID	Coccidioides immitis	Serology standard; high titers indicate dissemination
Candida Mannan/Anti-mannan	Candida spp.	Combined testing improves sensitivity

Antibody Detection (Precipitin Tests)

Immunodiffusion (ID): Detects precipitating antibodies (H and M bands for Histoplasma, A and B bands for Blastomyces)
Complement Fixation (CF): Quantitative; titer ≥1:32 suggests active disease or dissemination
Exoantigen test (historical): Fungal cultures treated with formalin; filtrate tested by immunodiffusion to confirm dimorphic fungi

6. Biochemical Tests (for Yeasts)

Test	Application
Germ tube test	Candida albicans produces germ tubes in serum at 37°C within 2-3 hours; rapid presumptive ID
Urease test	Cryptococcus (+), Trichosporon (+); Candida (-)
Cornmeal agar morphology	Chlamydoconidia of C. albicans; pseudohyphal patterns
Carbohydrate assimilation (API 20C AUX, Vitek 2)	Automated biochemical yeast identification

7. Molecular Methods

Increasingly used for rapid, definitive identification:

PCR-based assays: Detect fungal DNA directly in clinical specimens; useful for Aspergillus, Candida, Pneumocystis jirovecii
Matrix-Assisted Laser Desorption Ionization - Time of Flight (MALDI-TOF MS): Identifies fungi from isolated colonies within minutes by proteomic fingerprinting; excellent for yeasts, improving for molds
DNA sequencing (ITS region): Sequencing of the Internal Transcribed Spacer (ITS1/ITS2) region of rRNA gene - the gold standard for species-level identification of unusual isolates; D1/D2 region for yeasts
Next-Generation Sequencing (NGS) / metagenomic sequencing: For unidentified or unculturable organisms

8. Antifungal Susceptibility Testing

Once a fungal isolate is recovered, susceptibility testing guides therapy:

CLSI M27 (yeasts) and M38 (molds) broth microdilution methods are the reference standards
Minimum Inhibitory Concentration (MIC) is determined: the lowest drug concentration inhibiting visible growth
Interpretive breakpoints (Susceptible / Intermediate / Resistant) are established for many drug-fungus combinations
Commercial options: Etest strips (gradient diffusion), Sensitire YeastOne, Vitek 2 AST

Summary Table: Lab Diagnosis by Mycosis Category

Category	Key Methods
Superficial (e.g., pityriasis versicolor, tinea nigra)	KOH scraping, Wood's lamp fluorescence
Cutaneous (e.g., dermatophytosis, onychomycosis)	KOH, SDA culture at 25°C, hair perforation test
Subcutaneous (e.g., sporotrichosis, chromo-, mycetoma)	Biopsy, histology (GMS/PAS), culture, "asteroid bodies" or "sclerotic bodies"
Systemic/Endemic (e.g., histoplasmosis, coccidioidomycosis)	Urine antigen, serology (ID/CF), BSL-3 culture, bone marrow/biopsy
Opportunistic (e.g., candidiasis, aspergillosis, cryptococcosis)	Blood culture, CrAg, galactomannan, β-D-glucan, MALDI-TOF, PCR

Sources: Jawetz Melnick & Adelberg's Medical Microbiology 28th Ed.; Medical Microbiology 9e (Murray)

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