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Microbiology

Specimen Sources

By understanding the specific requirements for each specimen type and the corresponding laboratory procedures, you’ll be well-equipped to contribute to the accurate and timely diagnosis of fungal infections. This section covers the different specimen sources in mycological analysis: superficial, deep/systemic, and systemic and the key aspects of specimen collection and analysis for mycological testing

Superficial Mycoses

  • Location: Affect the outermost layers of the body – skin, hair, and nails
  • Common Pathogens: Dermatophytes (Trichophyton, Microsporum, Epidermophyton), Malassezia furfur
  • Specimen Sources
    • Skin Scrapings: Collected from the active edge of lesions
    • Hair Samples: Plucked hairs (with roots) or cut hairs from infected areas
    • Nail Clippings/Scrapings: Clippings from affected areas, scrapings from under the nail plate
  • Collection Techniques
    • Clean the area with 70% alcohol
    • Use sterile instruments (scalpel, curette, forceps)
    • Collect adequate material (scales, hair with roots, nail debris)
    • Store in a sterile, dry container
  • Key Lab Procedures
    • Direct Microscopic Examination
      • KOH preparation to dissolve keratin and visualize fungal elements
      • Calcofluor white stain for enhanced visualization under fluorescence microscopy
    • Culture
      • Inoculation onto Sabouraud Dextrose Agar (SDA) or Dermatophyte Test Medium (DTM)
      • Incubation at 25-30°C for up to 4 weeks
    • Identification
      • Based on colony morphology and microscopic characteristics
  • Diagnostic Goal: To identify the specific fungus causing the superficial infection, enabling targeted treatment with topical or oral antifungal medications

Deep and Systemic Mycoses

  • Location: Involve internal organs, tissues, and/or the bloodstream
  • Common Pathogens: Dimorphic fungi (Histoplasma, Blastomyces, Coccidioides), molds (Aspergillus, Fusarium), opportunistic yeasts (Candida, Cryptococcus)
  • Specimen Sources
    • Respiratory: Sputum, bronchial wash/lavage, bronchial brushings, lung biopsy, pleural fluid
    • Bone: Bone biopsy, bone marrow aspirate/biopsy
    • Tissue: Tissue biopsy (from affected organ), cerebrospinal fluid (CSF)
  • Collection Techniques
    • Strict aseptic technique is crucial
    • Collect from the most affected area of the tissue/organ
    • Use appropriate collection methods (bronchoscopy, surgical biopsy, lumbar puncture)
    • Transport specimens promptly to the lab
  • Key Lab Procedures
    • Direct Microscopic Examination
      • KOH preparation, Gram stain, GMS (Gomori Methenamine Silver) stain, PAS (Periodic Acid-Schiff) stain, India ink (for Cryptococcus), Calcofluor white
    • Culture
      • Inoculation onto SDA, Brain-Heart Infusion (BHI) agar, Inhibitory Mold Agar (IMA)
      • Incubation at 25-30°C and 35-37°C for up to 4 weeks
    • Identification
      • Based on colony morphology, microscopic characteristics, and molecular methods (PCR, sequencing)
    • Other Diagnostic Tests
      • Antigen detection (e.g., Histoplasma, Blastomyces, Cryptococcus, Galactomannan, Beta-D-Glucan assays)
      • Antibody detection (e.g., Coccidioides, Aspergillus antibodies)
      • Molecular detection (PCR) for rapid identification
      • Histopathology with special stains
  • Diagnostic Goal: To identify the specific fungus causing the deep or systemic infection, assess the extent of the infection, and guide appropriate antifungal therapy (often requiring intravenous or oral medications)

Systemic Mycoses (Blood and Bone Marrow Focus)

  • Location: Primarily focuses on infections detectable in the bloodstream (fungemia) and bone marrow
  • Common Pathogens: Candida, Aspergillus, Cryptococcus, Histoplasma, Coccidioides
  • Specimen Sources
    • Blood: Whole blood for culture, lysed blood, serum/plasma for antigen/antibody detection
    • Bone Marrow: Bone marrow aspirate and biopsy
  • Collection Techniques
    • Meticulous aseptic technique is critical to avoid contamination
    • Collect the appropriate volume of blood or bone marrow
    • Transport specimens to the lab promptly
  • Key Lab Procedures
    • Blood Culture
      • Automated blood culture systems with incubation for up to 5-7 days
      • Gram stain and fungal identification upon positive detection
    • Lysed Blood Culture
      • Lysis-centrifugation to concentrate fungi
      • Microscopic examination and culture of the sediment
    • Antigen Detection
      • Galactomannan, Beta-D-Glucan, Cryptococcal antigen, Histoplasma antigen assays
    • Molecular Detection (PCR)
      • Direct detection of fungal DNA in blood
    • Bone Marrow Analysis
      • Microscopic examination of smears and touch preparations
      • Histopathology with special stains (GMS, PAS)
      • Culture of aspirate and biopsy specimens
      • Molecular detection (PCR)
  • Diagnostic Goal: To rapidly detect the presence of fungi in the bloodstream or bone marrow, identify the specific pathogen, and guide prompt initiation of antifungal therapy

Key Considerations Across All Specimen Types

  • Aseptic Technique: Always use strict aseptic technique during collection
  • Adequate Volume: Collect an adequate amount of material for testing
  • Prompt Transport: Transport specimens to the lab promptly
  • Communication: Communicate with the lab regarding the patient’s clinical history and suspected pathogens
  • Safety: Handle specimens with appropriate precautions
  • Accurate Reporting: Report all results clearly and accurately, with interpretive comments
  • Antifungal Susceptibility Testing: Perform antifungal susceptibility testing on clinically significant isolates to guide therapy