Mycology & Virology Lab Diagno

Cheatsheet Content

### Specimen Collection Principles - Accurate diagnosis of mycotic infections relies on appropriate specimen collection. - Fungal and mycobacterial infections often present similarly, so specimens are frequently cultured for both. ### Respiratory Tract Specimens - **Samples:** Sputum, Induced Sputum, Bronchial Washings (BW), Bronchoalveolar Lavage (BAL), Tracheal Aspirate. - **Collection:** - Most common for fungal culture. - Patients should collect sputum from a deep cough in the morning. - **Note:** A nebulizer may be needed if the patient cannot produce sputum. - All sputum specimens must be collected in a sterile, screw-cap container. - **Transport:** Quickly; avoid delays. - **Processing:** - May be stored at room temperature if processed within 2 hours. - If delayed, refrigerate at 4°C. - Antimicrobial agents are used to prevent bacterial overgrowth. - **Note:** Upper respiratory tract has normal flora; use **0.5mL cycloheximide** (antifungal agent) in the medium. - If viscous, digest with **N-acetyl-L-cysteine** and concentrate. Use both nonselective and selective media with antimicrobials. Prepare **KOH mount**. ### Nasal Sinus Specimens (Surgical) - **Collection:** Collected surgically by ENT. - **Transport:** Immediately. - **Processing:** Plate directly onto media containing antimicrobials, **except cycloheximide** (inhibits some pathogenic molds). ### Vaginal Specimens - **Collection:** Use a sterile swab or fluid in a sterile container. - **Transport:** Within 24 hours of collection using culture transport swabs. - **Processing:** - Screen for yeasts and incubate at 30°C for 7 days. - Culture only when clinically indicated; direct smear microscopy is helpful. - **Note:** Always check the fungal form (yeast or mold) and temperature requirements (room temp or 35-37°C). ### Urine Specimens - **Collection:** - 24-hour urine samples are unacceptable. - A first voided morning urine specimen is preferred. - **Transport:** Promptly; may refrigerate briefly if delay is unavoidable. - **Processing:** - Process as soon as possible. - Centrifuge all urine samples; use the sediment (pellet) for culture. - **Note:** If a urine transport system is used, samples can be stored at room temperature for up to 72 hours. ### Cerebrospinal Fluid (CSF) - **Collection:** By physician, in a sterile screw-cap tube. Aseptically collect 25 mL for concentration. - **Transport:** Immediately at room temperature; **do not refrigerate**. - **Processing:** - Concentrated by centrifugation before inoculation (especially if sample 5mL, filter through a membrane filter (0.45µm pore size). Place filter portions on media. - Use 1 drop for **India ink** or **latex agglutination** (for *Cryptococcus*). - **Note:** CSF is sterile; **no antibiotics needed**. **Never refrigerate CSF specimens.** ### Blood Specimens - **Collection:** No special considerations. Collect 20-30 mL for adults; divide into 2 bottles. Maintain proper blood-to-broth ratio (1:5 to 1:10). - **Transport:** Room temperature; process immediately. - **Processing:** - **Lysis Centrifugation System:** Used for dimorphic fungi (e.g., *Histoplasma capsulatum*). - Most detected within 4 days; *H. capsulatum* recovery: 10-14 days. - **Automated Blood Culture System:** Provides accurate etiology determination. - **Isolator tube:** Most sensitive method for fungal recovery. - Optimal temperature for fungal blood cultures: **30°C**. Suggested incubation time: **21 days**. - **Note:** Systemic fungal infection should be incubated at **30°C**. ### Eye Specimens (Corneal Scrapings, Vitreous Humor) - **Collection:** By a physician. - **Processing:** - **Corneal scrapings:** Place directly onto microscopic slides and inoculate non-inhibitory media in X- or C-shaped patterns. - **Vitreous humor aspirate:** Concentrated by centrifugation (similar to CSF). ### Tissue, Bone Marrow, & Other Body Fluids - **Collection:** By a physician. - **Tissue (biopsy, surgical):** Collect fresh, unfixed tissue. Avoid formalin. Keep moist in a sterile container (e.g., small amount of sterile saline). Process promptly. - **Bone Marrow:** Collect a heparinized specimen. Transport immediately. - **Processing:** - **Tissue (biopsy, surgical):** Gently mince tissue; avoid grinding unless needed for KOH or calcofluor white prep (can destroy zygomycetes). Target purulent/discolored areas. Inoculate minced tissue on media. - **Heparinized bone marrow:** Plate directly onto media at the bedside. - **Note:** Blood culture bottles are not recommended. ### Dermatophyte Specimens: Hair - **Collection:** Infected hairs are removed by plucking with forceps. - **Processing:** - **Wood lamp:** Emits UV light (>365nm) to identify infected hairs. - **Mycosel agar** with chloramphenicol and cycloheximide for satisfactory recovery. - **Note:** If *Malassezia furfur* suspected, supplement media with **olive oil**. ### Dermatophyte Specimens: Skin (Scrapings) - **Collection:** - Clean skin with **70% isopropyl alcohol** before sampling. - Scrape using a scalpel blade or microscopic slide from the outer edge of a surface lesion. - Keep dry in a sterile container; process quickly to prevent contaminant overgrowth. - **Processing:** - **10% KOH wet mount:** Prepared with scrapings. - **Note:** KOH breaks down tissue, making fungal hyphae easier to view. - Use **Mycosel agar** with chloramphenicol and cycloheximide. ### Dermatophyte Specimens: Nails (Scrapings/Cuttings) - **Collection:** - Submitted as scrapings, cuttings, or occasionally a complete nail. Clean with **70% isopropyl alcohol**. - Use sterile scissors to cut complete nails into small thin strips. - Keep dry, in sterile screw-cap container. Immediate transport required. - **Processing:** - **10% KOH preparation.** - **Note:** Hair, skin, and nails should be placed in a sterile container; **do not refrigerate**. - Use selective media to suppress bacteria and rapid molds. - Use **Mycosel agar** with chloramphenicol and cycloheximide. ### Other Specimens: Feces - **Collection:** Collect in sterile container; usually for bacterial studies but yeasts may appear. - **Transport:** Quickly; do not allow drying. - **Processing:** If fungal culture requested: examine microscopically and inoculate sediment after concentration. ### Other Specimens: Abscess Fluid / Wound Exudates - **Collection:** Aspirate or collect exudate into sterile container; inspect for granules (indicative of mycetoma). - **Transport:** Immediately to avoid contaminant overgrowth. - **Processing:** If granules present, process separately. Plate material directly. ### Predominant Culture Sites for Recovery of Causative Agents | Infection | Respiratory | Blood | Bone Marrow | Tissue | Skin | Mucus | Bone | |----------------------|-------------|-------|-------------|--------|------|-------|------| | Blastomycosis | + | | | + | | | | | Histoplasmosis | + | + | + | + | | | | | Coccidioidomycosis | + | | | + | + | | | | Paracoccidioidomycosis| + | | | + | | | | | Sporotrichosis | | | | + | + | | | | Chromoblastomycosis | | | | + | + | | | | Eumycotic mycetoma | | | | + | | | + | | Phaeohyphomycosis | | | | + | + | | | *Note: Organisms may be recovered from multiple sites in disseminated infections.* ### Clinical Specimen Flowchart ```mermaid graph TD A[Clinical Specimen] --> B{Direct Examination}; A --> C{Culture}; B --> D[Cerebrospinal Fluid]; B --> E[Skin, Hair, Nails, Other Selected Specimens]; D --> F[India Ink]; D --> G[Cryptococcal Antigen]; E --> H[KOH or KOH with Calcofluor White]; H --> I{Hyphal Elements}; I --> J[Width]; I --> K[Hyaline vs. Dematiaceous]; I --> L[Septate vs. Sparsely Septate]; I --> M[No Hyphal Elements]; C --> N[Tissue]; C --> O[Blood]; N --> P[Histology Stain]; N --> Q[Fluorescent Antibody (selected organisms)]; C --> R{Growth at 22-30°C}; C --> S{Growth at 35°C (suspect dimorphic)}; R --> T[Yeast]; R --> U[Mold]; S --> V[No Growth]; T --> W[Special Supplementary Media]; W --> X[Germ Tube]; W --> Y[Biochemicals]; Y --> Z[Assimilation]; Y --> AA[Fermentation]; X --> BB[Cornmeal]; BB --> CC[Blastoconidia]; BB --> DD[Chlamydospores]; X --> EE[Other]; EE --> FF[Birdseed]; EE --> GG[Caffeic Acid]; EE --> HH[Urea]; U --> II[Microscopic (LPCB)]; U --> JJ[Macroscopic]; II --> KK[Conidia]; KK --> LL[Size]; KK --> MM[Shape]; KK --> NN[Arrangement]; II --> OO[No Conidia]; II --> PP[Slide Culture]; PP --> QQ[Hyphae]; QQ --> RR[Hyaline vs. Dematiaceous]; QQ --> SS[Septate vs. Sparsely Septate]; QQ --> TT[Rhizoids]; JJ --> UU[Color/Pigment]; JJ --> VV[Texture]; JJ --> WW[Growth Rate]; ```