Procedures

This section covers the key aspects of analytic procedures in mycology, covering specimen sources, major pathogens, identification methods, and antifungal susceptibility testing

Specimen Sources

• Superficial: Skin scrapings, hair samples, nail clippings/scrapings
• Deep and Systemic
  • Respiratory: Sputum, BAL, bronchial washings/brushings, lung biopsy, pleural fluid
  • Bone: Bone biopsy, bone marrow aspirate/biopsy
  • Tissue: Tissue biopsy (from affected organ), CSF, blood
• Systemic (Blood and Bone Marrow Focus): Blood (whole blood, lysed blood, serum/plasma), bone marrow aspirate/biopsy

Major Pathogens and Disease States

• Yeasts
  • Candida: Candidiasis (thrush, vaginitis, invasive candidiasis)
  • Cryptococcus: Meningitis, pneumonia
  • Malassezia: Pityriasis versicolor, folliculitis
• Dimorphic Fungi
  • Histoplasma: Histoplasmosis (pulmonary, disseminated)
  • Blastomyces: Blastomycosis (pulmonary, cutaneous, disseminated)
  • Coccidioides: Coccidioidomycosis (pulmonary, disseminated)
  • Sporothrix: Sporotrichosis (cutaneous, lymphocutaneous)
• Dermatophytes: Trichophyton, Microsporum, Epidermophyton (Tinea infections)
• Mucorales (Zygomycetes): Rhizopus, Mucor (Mucormycosis)
• Hyaline Molds
  • Aspergillus: Aspergillosis (invasive, aspergilloma, ABPA)
  • Fusarium: Keratitis, onychomycosis, disseminated infections
  • Penicillium: Penicilliosis, rare infections
  • Scedosporium: Mycetoma, sinusitis, disseminated infections
• Dematiaceous Molds: Alternaria, Cladosporium, Fonsecaea (Skin infections, sinusitis, chromoblastomycosis)
• Pneumocystis jirovecii: PCP (Pneumocystis pneumonia)
• Microsporidium spp.: Microsporidiosis (intestinal, keratoconjunctivitis, disseminated)

Colony Morphology and Growth Characteristics

• Key Features: Growth rate, texture, elevation, margin, color (top and reverse), other features (exudate, diffusible pigment, zonation)
• Temperature and Incubation Time: Most fungi grow at 25-30°C for up to 4 weeks
• Examples: (See detailed sections for specific pathogens)
  • Candida: Creamy, white colonies
  • Aspergillus: Velvety/granular, blue-green colonies
  • Rhizopus: Rapid growth, cottony colonies

Microscopic Identification

• Key Structures: Hyphae (septate vs. non-septate, hyaline vs. dematiaceous), conidia (microconidia, macroconidia, arthroconidia), yeast cells, spherules, sclerotic bodies, etc
• Staining Techniques: KOH prep, Gram stain, GMS stain, PAS stain, India ink, calcofluor white
• Examples: (See detailed sections for specific pathogens)
  • Aspergillus: Septate hyphae with dichotomous branching
  • Coccidioides: Spherules with endospores
  • Rhizopus: Broad, non-septate hyphae with rhizoids

Direct Antigen and Molecular Detection

• Direct Antigen Detection
  • Techniques: EIA, latex agglutination, lateral flow immunoassay
  • Examples: Cryptococcal antigen (CrAg), galactomannan (Aspergillus), beta-D-glucan, Histoplasma antigen, Blastomyces antigen
• Molecular Detection
  • Techniques: PCR, real-time PCR, multiplex PCR, sequencing
  • Examples: Pneumocystis jirovecii PCR, Aspergillus PCR, Candida PCR, panfungal PCR

Other Identification Methods

• Biochemical Tests: Carbohydrate assimilation/fermentation, urease, germ tube test, etc
• Automated Methods: Vitek 2, MicroScan, Sensititre
• MALDI-TOF MS: Rapid and accurate protein profiling

Antifungal Susceptibility Testing (AFST)

• Manual Methods
  • Broth Microdilution (BMD): Gold standard
  • Etest: Easy to perform
• Automated Methods: Streamline testing, faster turnaround
• Classes of Antifungal Agents
  • Azoles: Inhibit ergosterol synthesis (fluconazole, voriconazole, etc.)
  • Polyenes: Disrupt cell membrane (amphotericin B)
  • Echinocandins: Inhibit beta-1,3-glucan synthesis (caspofungin, etc.)
  • Allylamines: Inhibit squalene epoxidase (terbinafine)
  • Flucytosine (5-FC): Inhibits DNA/RNA synthesis
  • Griseofulvin: Disrupts microtubule function
• Intrinsic Resistance Patterns
  • Candida krusei: Fluconazole
  • Candida glabrata: Reduced azole susceptibility
  • Scedosporium spp.: Amphotericin B, many azoles
  • Mucorales: Voriconazole