Laboratory Diagnosis of Fungal Infections

Laboratory Diagnosis of Fungal Infections

Laboratory Diagnosis of Fungal Infections

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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. Microscopy of budding yeast and pseudohyphae

StainUseFungi (Examples)Key Finding
$10-20% KOH## Direct Microscopic Examination - Scope The Spores
Rapid, cost-effective initial detection. Observe: yeast, pseudohyphae, true hyphae (septate/aseptate), spores, spherules.
Microscopy of budding yeast and pseudohyphae

| 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:

MediumHighlightsUse
SDADextrose, PeptoneGeneral 📌 Sweet Dreams for Aspergillus
Mycosel/MycobioticSDA + Abx + CycloheximidePathogens (inhibits contaminants)
BHI AgarBrain Heart InfusionFastidious, dimorphic yeast
Caffeic Acid AgarPhenol oxidase detectionC. neoformans (melanin)

Identification:

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

Penicillium marneffei colony and LPCB mount microscopy

⭐ 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

Test (Antigen/Ab)Target Fungus/MarkerSampleSignificance/Limitations (Common Methods)
Galactomannan (Ag)Aspergillus spp.Serum, BALIndex >0.5 for IA; false positives. (ELISA)
(1→3)-β-D-glucan (BDG) (Ag)Pan-fungal (not Mucorales, Crypto, Blasto)SerumInvasive fungal infection marker; non-specific. (ELISA)
Cryptococcal Ag (CrAg)C. neoformans/gattii (capsular Ag)CSF, SerumDiagnosis/prognosis of cryptococcosis. (Latex Agglutination, LFA, EIA)
Antibody (Ab) DetectionHistoplasma, Coccidioides, BlastomycesSerumEndemic mycoses; less useful in immunocompromised. (ID, CF)

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 vs Mucor hyphae in histopathology

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.

Practice Questions: Laboratory Diagnosis of Fungal Infections

Test your understanding with these related questions

A 10-year-old boy presented with painful boggy swelling of scalp, multiple sinuses with purulent discharge, easily pluckable hair, and lymph nodes enlarged in occipital region, which of the following would be most helpful for diagnostic evaluation?

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Flashcards: Laboratory Diagnosis of Fungal Infections

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_____ is used to isolate fungi

Hint: Which special culture media/agar

TAP TO REVEAL ANSWER

_____ is used to isolate fungi

Sabouraud agar

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