Laboratory Diagnosis of Fungal Diseases Reviewer

Laboratory Diagnosis of Fungal Diseases — Reviewer

I. SAFETY ISSUES

• Use Class II or higher biosafety cabinets when handling cultures

• Use enclosed electric incinerator for loops/needles

• Use screw-capped tubes/slants for filamentous fungi; seal Petri dish edges with Parafilm/tape

• Flood plates with 4%–10% formalin for several hours before microscopic examination

II. SPECIMEN COLLECTION, HANDLING & TRANSPORT

• Collect appropriate specimens — primary criterion for accurate diagnosis

• Process specimens ASAP (fungi grow slowly; delay = contaminant overgrowth)

• Reject unsatisfactory or improperly labeled specimens

• Primary isolation medium must contain antimicrobial agents for contaminated specimens (hair, skin, nails)

Predominant Culture Sites by Infection:

III. PROCESSING OF SPECIMENS

1. Blood & Bone Marrow Aspirate

• Volume: 20–30 mL blood (adults), divided into two bottles

• Dilution: 5- to 10-fold with broth medium

• Culture systems:

  • Direct culture — centrifuge, inoculate buffy coat (0.5–1 mL) onto fungal media

  • Lysis centrifugation isolator — lyses blood cells, concentrates fungi, plates sediment

  • Automated systems (BACTEC) — detects fungal growth via CO₂ production

• Initial smears: Giemsa, Gram, PAS stains

• Remaining sample: 3–5 mL; inoculate onto SDA, BHIA, or biphasic systems

• Primary media: SDA + chloramphenicol + gentamicin (26°C & 35°C); BHIA + 5% sheep blood (35°C)

• Maintain cultures for 4 weeks

2. CSF

• Volume: 3–5 mL; do NOT refrigerate (room temp or 30°C)

• Centrifuge → supernatant for cryptococcal antigen testing (LAT)

• Sediment for India ink mount or EIA/LFA

• Culture on SDA-CG and BHIA + 5% sheep blood; maintain 4 weeks

3. Respiratory Tract Specimens

• Best collected in the morning; use 2 sterile Dacron swabs

• Store at 4°C if short delays

• Sputa: emulsify with glass beads or digest with N-acetyl-L-cysteine

• Prep: 10% KOH wet mount + Gram-stained smear (PAS if KOH unsatisfactory)

• Culture: SDA-CG (26°C & 35°C) + BHIA + 5% sheep blood (35°C); maintain 4 weeks

4. Urine

• Midstream, clean-catch, or catheterized (best); process within 2 hours or refrigerate

• Centrifuge (10–15 min, 2000 rpm); smear and culture sediment

• KOH mount; inoculate 0.05–0.1 mL onto SDA-CG; maintain 4 weeks

5. Vaginal & Cervical Discharges

• 2 sterile swabs; place in transport media

• KOH mount + Gram-stained smear; culture on SDA-CG; incubate at 26°C & 35°C; maintain 4 weeks

6. Skin Scrapings, Hair & Nails

• Clean area with 70% alcohol first

• Hair — pluck by roots; select fluorescing/broken/scaly hair (Wood lamp, 365 nm)

• Nail — scrape discolored areas; collect keratin debris; cut into small pieces

• Skin — firmly scrape advancing border of lesion

• Direct microscopy: 10%–20% KOH and/or Calcofluor white

• Culture on SDA + cycloheximide + chloramphenicol + gentamicin (DERMASEL) at 26°C, 4 weeks

7. Tissues

• Collect aseptically; keep moist with sterile saline/BHI broth; portion in formalin for histopathology (H&E, GMS, PAS)

• Include normal tissue AND center & edge of lesion; tease apart, mince/homogenize

• Check for granules; note color

8. Wound Exudates, Plaques, Abscess Fluid

• Scrape plaque (Candida); needle aspiration of deep cysts/abscesses

• Check for granules; note color

IV. DIRECT MICROSCOPIC EXAMINATION

Purposes:

1. Rapid report → early treatment initiation

2. Morphologic clues to genus of organism

3. Evidence of infection despite negative culture (patients on antifungal therapy)

Methods:

Staining Characteristics:

Other stains: Gram, Acid-fast (Nocardia), Fluorescent auramine-rhodamine, Papanicolaou, Fontana-Tribondeau, Griffith's (Grocott)

V. ISOLATION / CULTURE METHODS

General Conditions:

• Media must include N source (amino acids/urea), C source (glucose)

• With or without antimicrobial agents (cycloheximide, gentamicin, chloramphenicol, ciprofloxacin)

• Aerobic; 40–50% humidity

• Examine growth 3x weekly up to 4 weeks

• 26–30°C for molds; 35–37°C for yeasts

Container Comparison:

Primary Media:

Selective/Differential Media:

Incubation Guidelines:

• Routine: room temp or 30°C

• Dimorphic: 35°C

• Duration: 4–6 weeks, examine twice weekly

• Fast growers: Mucor, Rhizopus (days); Slow growers: Fonsecaea, Phialophora (2 weeks)

VI. MICROSCOPIC EXAMINATION OF CULTURES

Methods:

• Tease mount-LPCB — remove mycelium from middle third of colony; place in LPCB drop; examine at low and high magnification

• Cellophane tape/flag prep — touch sticky side to colony surface; place on LPCB drop; read within 30 minutes; no coverslip needed

• Slide culture (Riddel's technique) — demonstrates natural morphology; encourages conidiation; can be preserved long-term; requires more skill and time

Macroscopic Observations:

• Rate of growth (rapid/slow)

• Topography (flat, rugose, umbonate, verrucose)

• Texture (cottony, velvety, powdery, granular, creamy)

• Pigmentation (surface & reverse)

Microscopic Observations:

• Septate vs. aseptate hyphae

• Hyaline vs. phaeoid hyphae

• Reproductive structures

• Type, size, shape, arrangement of conidia/ascospores

VII. MISCELLANEOUS IDENTIFICATION TESTS

For Molds:

For Yeasts:

VIII. SURROGATE MARKERS

IX. ANTIFUNGAL SUSCEPTIBILITY TESTING (AFST)

• Standards: CLSI (Philippines) and EUCAST

  • CLSI M27-A4 — yeasts

  • CLSI M38-A3 — molds

  • CLSI M44 — disk diffusion for yeasts

  • CLSI M51 — disk diffusion for molds

• Broth microdilution — gold standard

• Limitation: breakpoints (MIC thresholds) not fully established for all fungi

Commercial AFST Systems:

Key mnemonics to remember:

• SDA-CG = Sabouraud Dextrose Agar + Chloramphenicol + Gentamicin

• BHIA = Brain Heart Infusion Agar

• LPCB = Lactophenol Cotton Blue

• AFST = Antifungal Susceptibility Testing

• Cultures maintained 4 weeks for most specimens; up to 6 weeks for some