Name: Emmanuelle Ionnie G.

Arcilla Subject: MLS 124: Mycology and Virology

Section: 3A Date: April 23, 2025

Laboratory Diagnosis of Fungal Infections

1. Safety Considerations
●​ Biological Safety Cabinet (Class II): Essential for handling fungal cultures to prevent
airborne conidia exposure.
●​ Use of Screw-Capped Tubes: Preferred over Petri dishes to minimize contamination
and dehydration.
●​ Incinerators: Electric incinerators are recommended to safely sterilize loops/needles.

2. Specimen Collection and Handling

●​ Types of Specimens: Hair, skin, nails, blood, bone marrow, CSF, respiratory secretions,
tissue, abscess fluid, urine, and feces.
○​ Hair/Skin/Nails: Clean with 70% alcohol; use Wood’s lamp for fluorescent fungi
(e.g., Microsporum).
○​ Blood/Bone Marrow: Lysis-centrifugation (Isolator tube) is most sensitive for
fungemia.
○​ CSF: Centrifuge for India ink (for Cryptococcus) or latex agglutination
(cryptococcal antigen).
○​ Respiratory Specimens: Induced sputum or bronchoalveolar lavage for
dimorphic fungi (e.g., Histoplasma).
○​ Tissue: Minced (not ground) to preserve hyphal structures.
●​ Specimen Transport: Sterile containers; refrigerate (4°C) if delayed processing
(except blood cultures).

3. Direct Microscopic Examination

●​ KOH Preparation (10–20%)
○​ Target: Hyphae/pseudohyphae
○​ Dissolves keratin to reveal hyphae or yeast
○​ Clinical use: Dermatophytes, Candida in skin/nails
●​ Calcofluor White:
○​ Target: Chitin (all fungi)
○​ Fluorescent stain binds chitin (fungi glow apple-green under UV)
○​ Clinical use: Rapid screening (fluorescence microscopy)
●​ India Ink:
○​ Target: Cryptococcus capsule
○​ Detects Cryptococcus neoformans capsules in CSF
○​ Clinical use: CSF analysis (now largely replaced by antigen tests)
●​ Histologic Stains:
○​ GMS (Gomori Methenamine Silver): Stains fungal cell walls black.
○​ PAS (Periodic Acid-Schiff): Stains polysaccharides pink.
○​ Giemsa: Identifies intracellular Histoplasma or Pneumocystis.
○​ Fontana-Masson: Detects melanin in phaeoid fungi.

4. Culture Media and Incubation

●​ Primary Media:
○​ Sabouraud Dextrose Agar (SDA): General-purpose; Emmons modification
(neutral pH) is preferred. Some Candida spp. grow poorly.
 ○​ SDA with Antibiotics: Chloramphenicol (bacteria) ± cycloheximide (contaminant
molds). Avoid for Cryptococcus
○​ Brain Heart Infusion (BHI) Agar: Enriched for dimorphic fungi (e.g.,
Histoplasma at 37°C). Slow growth (2–4 weeks)
○​ Chromogenic Agar (e.g., CHROMagar): Differentiates Candida species by
colony color. Costlier than SDA
○​ Birdseed Agar: Selective for C. neoformans/gattii. Requires 48–72h incubation.
●​ Incubation:
○​ 22–30°C: For mold phase of dimorphic fungi and most saprobes.
○​ 37°C: For yeast phase of dimorphic fungi (e.g., Blastomyces, Histoplasma).
○​ Duration: 4–6 weeks for slow growers (e.g., Histoplasma); Mucorales grow in
1–3 days.

5. Identification of Fungi
●​ Macroscopic Features: Colony texture (powdery, velvety), color (hyaline vs. phaeoid),
and growth rate.
●​ Microscopic Techniques:
○​ Tease Mount (LPCB): Disrupts conidia but stains hyphae.
○​ Cellophane Tape Prep: Preserves conidial arrangement (temporary).
○​ Slide Culture: Best for observing intact fruiting structures (e.g., Aspergillus
conidiophores).
●​ Key Morphologic Features:
○​ Hyphae: Septate (e.g., Aspergillus) vs. sparsely septate (e.g., Mucorales).
○​ Conidia: Arrangement (e.g., Aspergillus phialides, Fusarium macroconidia).
○​ Dimorphism: Mold (25°C) vs. yeast/spherule (37°C; e.g., Histoplasma,
Coccidioides).

6. Special Tests for Yeasts

●​ Germ Tube Test: Candida albicans and C. dubliniensis produce germ tubes in serum (3
hours, 37°C).
●​ Cornmeal Agar: Differentiates Candida by chlamydospores, pseudohyphae, or true
hyphae.
●​ Biochemical Tests:
○​ Carbohydrate Assimilation (API 20C): Identifies sugars utilized (e.g., C.
glabrata ferments trehalose).
○​ Urease: Cryptococcus (+) vs. Candida (−).
○​ Potassium Nitrate Assimilation: Cryptococcus spp. differentiation.
●​ Chromogenic Media: CHROMagar for presumptive ID of C. albicans (green), C.
tropicalis (blue), C. krusei (pink).
●​ Temperature Studies: Differentiate yeast species based on thermotolerance (critical for
Candida and Cryptococcus).

Organism 25–30°C 35–37°C 42–45°C Key Clinical

Utility

Cryptococcus Optimal Weak/slow No growth Rules out

spp. growth Cryptococcus
if growth at
42°C

C. albicans Growth Optimal Growth Confirms ID

(vs. C.
dubliniensis)
C. dubliniensis Growth Growth No growth Distinguishes
from C.
albicans

C. glabrata Growth Growth Variable May indicate

azole
resistance

Thermophilic Growth Optimal Growth (some Aspergillus

molds spp.) fumigatus
(tolerates 50°C)

Integration with Other Tests

1.​ Germ Tube (+) + Growth at 45°C = C. albicans.

2.​ Urease (+) + No growth at 42°C = Cryptococcus.
3.​ Chromogenic agar + Thermotolerance = Rapid Candida speciation.

7. Immunodiagnosis and Antigen Detection

●​ Cryptococcal Antigen (Latex Agglutination/ELISA): Gold standard for Cryptococcus
in CSF/serum.
●​ (1-3)-β-D-Glucan Assay (Fungitell): Detects most fungi (except Mucorales); useful for
invasive infections.
○​ False Positives: Hemodialysis, bacterial infections.
●​ Galactomannan ELISA: For Aspergillus (high sensitivity in neutropenic patients).
●​ Antibody Tests: Limited utility due to cross-reactivity (e.g., Histoplasma
immunodiffusion).
●​ Cryptococcal Ag (LA/EIA): For C. neoformans/gattii. Gold standard for meningitis.

8. Antifungal Susceptibility Testing (AST)

●​ Common Antifungals:
○​ Polyenes (Amphotericin B): Broad-spectrum but nephrotoxic.
○​ Azoles (Fluconazole, Voriconazole): Yeasts (FLU) vs. molds (VORI/ITRA).
○​ Echinocandins (Caspofungin): For Candida and Aspergillus.
●​ CLSI M27/M38: Broth microdilution for yeasts/molds.
●​ Resistance Concerns: Candida glabrata (FLU resistance), Aspergillus (emerging azole
resistance).

9. Diagnostic Challenges
●​ Dimorphic Fungi: Require conversion to yeast phase (37°C) for confirmation (e.g.,
Histoplasma).
●​ Contaminants vs. Pathogens: Clinical correlation is critical (e.g., Aspergillus in sputum
vs. tissue invasion).
●​ Rare/Novel Pathogens: Molecular methods (PCR, sequencing) may be needed for
unusual isolates.