Laboratory Diagnosis of Fungal Infections

Specimen Collection & Transport - Fungal Haul & Handle

• Specimens: Skin, nails, hair; sputum, CSF, blood, tissue.
• Collection: Aseptic technique into sterile containers.
• Site Selection: Crucial; e.g., active lesion edge for skin.
• Quantity: Adequate for microscopy, culture, molecular tests.
• Transport: Rapid, at appropriate temperature (often room temp), prevent drying.

  ⭐ For dermatophytes, scrape the active, erythematous border of skin lesions.

Direct Microscopic Examination - Scope The Spores

Rapid, cost-effective initial detection. Observe: yeast, pseudohyphae, true hyphae (septate/aseptate), spores, spherules.

| Clears tissue; 📌 KOH Kills Our Host cells, Fungi Freely Seen | Most fungi | Fungal elements | | India Ink | Capsule stain | Cryptococcus neoformans | Halo around yeast | | Calcofluor White | Chitin binding, fluorescence | Most fungi | Apple-green fluorescence | | Gram Stain | Yeast stain | Candida spp. | Gram +ve yeasts, pseudohyphae | | Giemsa/Wright | Intracellular fungi | Histoplasma capsulatum | Yeasts in macrophages |> ⭐ Calcofluor white stain requires a fluorescence microscope and binds non-specifically to cellulose and chitin.

Fungal Culture - Grow & Glow Fungi

Definitive ID, speciation & susceptibility.

Culture Media & Incubation:

Identification:

• Macroscopic: Colony (texture, color, pigment).
• Microscopic: LPCB (hyphae, conidia, spores).
• Germ Tube Test: C. albicans (+ve 2-3h, 37°C).

⭐ Dimorphic fungi exhibit thermal dimorphism: mold form at 25-30°C (saprophytic/environmental) and yeast form at 37°C (pathogenic/in vivo).

Serology & Antigen Detection - Immune Clue Hunt

Molecular Methods - DNA Fungal Fingerprints

• PCR: Conventional, Real-time (qPCR), Multiplex.
  • Targets: Internal Transcribed Spacer (ITS) regions, 18S/28S rRNA genes.
• Advantages: ↑Sensitivity, ↑Specificity, rapid Turn-Around Time (TAT); detects non-viable/fastidious fungi.
• Other Techniques:
  • Probes & Sequencing: For speciation, phylogeny.
  • MALDI-TOF MS: Rapid identification of yeast/mold from culture.
  • PNA-FISH (Peptide Nucleic Acid Fluorescence In Situ Hybridization).

⭐ Internal Transcribed Spacer (ITS) regions of ribosomal DNA are common targets for fungal PCR due to interspecies variability and conserved flanking regions for primer binding an ITS region is a piece of non-functional RNA situated between structural ribosomal RNAs on a common precursor transcript

Histopathology - Tissue Tales: Fungi

• Biopsy vital for invasive infections: shows fungi & host response.
• Key Stains: | Stain | Stains | Color | Uses | |---|---|---|---| | H&E | Host, morphology | Variable | Candida | | GMS | Walls | Black | General | | PAS | Glycogen | Magenta | General | | Mucicarmine | Capsule | Pink/Red | Cryptococcus | | Fontana-Masson | Melanin | Brown/Black | Dematiaceous, Crypto |* 📌 GMS: Great Myco Staining; PAS: Pretty Awesome Sugar.
• Morphology: Yeast, hyphae (septation, branching, pigment), spherules.

⭐ Aspergillus: septate hyphae, 45° branching. Mucorales: broad, non-septate/pauciseptate hyphae, wide-angle branching.

High‑Yield Points - ⚡ Biggest Takeaways

• KOH wet mount: For rapid screening and visualization of fungal elements in clinical samples.
• India Ink stain: Specifically demonstrates the polysaccharide capsule of Cryptococcus neoformans, especially in CSF.
• Sabouraud Dextrose Agar (SDA): The most common selective medium for fungal culture, often with antibiotics.
• Calcofluor White stain: A fluorescent stain binding to chitin and cellulose in fungal cell walls, enhancing detection.
• Serological tests (e.g., Galactomannan, Beta-D-Glucan) and PCR: Crucial for early diagnosis of invasive fungal infections.