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Microbiology

Deep & Systemic

Deep and systemic fungal infections pose significant diagnostic challenges due to their invasive nature and the difficulty in accessing affected tissues. Accurate identification of the causative agent is crucial for effective treatment, necessitating meticulous specimen collection and a comprehensive range of analytic procedures. This review focuses on the collection and processing of respiratory, bone, and tissue specimens for mycological analysis, highlighting the importance of proper technique, appropriate culture methods, and advanced diagnostic tools for the timely detection and identification of fungal pathogens in these critical clinical settings

Deep and Systemic Mycoses: An Overview

  • Severity: These infections are far more serious than superficial ones, as they involve internal organs, tissues, and even the bloodstream
  • Causative Agents: We’re often dealing with dimorphic fungi (like Histoplasma, Blastomyces, Coccidioides), molds (like Aspergillus, Fusarium), and opportunistic yeasts (like Candida and Cryptococcus in immunocompromised individuals)
  • Diagnostic Importance: Accurate and timely diagnosis is essential for initiating appropriate antifungal therapy and improving patient survival

Specimen Types and Collection Techniques

Respiratory Specimens

  • Purpose: To diagnose fungal pneumonia, invasive pulmonary aspergillosis, and other respiratory mycoses
  • Specimen Types
    • Sputum
      • Collection: Collect a deep cough specimen, not just saliva. First morning specimens are ideal. Rinse the mouth with water before collection to reduce bacterial contamination
      • Quality: Look for purulent (pus-containing) material. A poor-quality sputum sample (mostly saliva) is not suitable for fungal culture
    • Bronchial Wash/Lavage
      • Collection: Obtained during bronchoscopy. A sterile saline solution is instilled into the airways and then collected
      • Advantages: More representative of lower respiratory tract infections than sputum, especially in patients who can’t produce sputum
    • Bronchial Brushings
      • Collection: A brush is passed through the bronchoscope to collect cells and debris from the airway walls
      • Advantages: Can be useful for diagnosing localized infections
    • Lung Biopsy
      • Collection: Obtained via surgical or percutaneous (through the skin) methods. A small piece of lung tissue is removed for analysis
      • Advantages: Provides the most definitive diagnosis, especially in cases where other specimens are non-diagnostic
    • Pleural Fluid
      • Collection: Aspirated from the pleural space (between the lung and chest wall) via thoracentesis
      • Purpose: To detect fungal infections of the pleura (empyema)

Bone Specimens

  • Purpose: To diagnose fungal osteomyelitis (bone infection)
  • Specimen Types
    • Bone Biopsy
      • Collection: The gold standard. A piece of bone is removed surgically or via needle biopsy
      • Technique: Collect from the affected area of the bone. If possible, include both bone and adjacent soft tissue
    • Bone Marrow Aspirate/Biopsy
      • Collection: A sample of bone marrow is aspirated or a core of bone marrow tissue is removed
      • Purpose: Useful for diagnosing disseminated fungal infections that involve the bone marrow (e.g., histoplasmosis, coccidioidomycosis)

Tissue Specimens

  • Purpose: To diagnose fungal infections in various organs and tissues (e.g., liver, spleen, brain, skin)
  • Specimen Types
    • Tissue Biopsy
      • Collection: Obtained surgically or via needle biopsy. The specific technique depends on the location of the infected tissue
      • Considerations
        • Collect from the most affected area of the tissue
        • Include both necrotic (dead) and viable (living) tissue
        • If possible, submit multiple samples from different areas of the lesion
    • Cerebrospinal Fluid (CSF)
      • Collection: Collected via lumbar puncture (spinal tap)
      • Purpose: To diagnose fungal meningitis (e.g., cryptococcal meningitis)
    • Blood
      • Collection: Collected via venipuncture
      • Purpose: To detect disseminated fungal infections (fungemia) such as candidemia, aspergillosis, or fusariosis

General Considerations for Deep/Systemic Specimens

  • Aseptic Technique: Always use strict aseptic technique during collection to minimize the risk of bacterial or fungal contamination
  • Quantity: Collect an adequate amount of material. These infections can be difficult to diagnose, so a larger sample increases the chances of detection
  • Transport: Transport specimens to the lab promptly. If there’s a delay, follow the lab’s specific storage instructions (usually refrigeration)
  • Communication: Communicate with the lab regarding the suspected fungal pathogen and any relevant patient history (e.g., travel, immune status)
  • Safety: Handle specimens with appropriate precautions, as some fungi (e.g., Coccidioides) are highly infectious in the lab

Laboratory Analysis

Direct Microscopic Examination

  • KOH Preparation: Useful for visualizing fungal elements in tissue samples
  • Gram Stain: Can reveal the presence of fungal organisms, although it’s not as specific as other stains
  • GMS (Gomori Methenamine Silver) Stain: Stains fungal cell walls black, providing excellent contrast
  • PAS (Periodic Acid-Schiff) Stain: Stains fungal cell walls magenta
  • India Ink: Used to detect capsules around Cryptococcus neoformans in CSF
  • Calcofluor White: A fluorescent dye that binds to chitin in fungal cell walls, enhancing visualization under a microscope
  • Wright-Giemsa Stain: Used to detect Histoplasma capsulatum within macrophages in bone marrow or blood smears

Culture

  • Media
    • Sabouraud Dextrose Agar (SDA): A general-purpose fungal medium
    • Brain-Heart Infusion (BHI) Agar: Enriched medium for fastidious fungi
    • Inhibitory Mold Agar (IMA): Contains antibiotics to inhibit bacterial growth
  • Incubation
    • Incubate cultures at appropriate temperatures (25-30°C and 35-37°C) for up to 4 weeks
    • Some fungi (e.g., Histoplasma) may require longer incubation periods
  • Identification
    • Identify fungal isolates based on macroscopic (colony morphology) and microscopic characteristics (e.g., hyphal structure, conidia)
    • Molecular methods (e.g., PCR, sequencing) are increasingly used for rapid and accurate identification

Other Diagnostic Tests

  • Antigen Detection
    • Detects specific fungal antigens in serum, CSF, or urine
    • Examples: Histoplasma antigen, Blastomyces antigen, Cryptococcus antigen
  • Antibody Detection
    • Detects antibodies against specific fungi in serum
    • Examples: Coccidioides antibodies, Aspergillus antibodies
  • Molecular Detection (PCR)
    • Detects fungal DNA in clinical specimens
    • Highly sensitive and specific
    • Can be used to identify fungi directly from clinical specimens, even when culture is negative
  • Histopathology
    • Examination of tissue samples under a microscope to identify fungal elements and assess the host’s immune response
    • Special stains (e.g., GMS, PAS) are used to highlight fungal organisms

Reporting

  • Report all positive and negative results clearly and accurately
  • Include the type of specimen, the methods used, and the identification of any fungi isolated
  • Report any relevant information about the specimen quality or the presence of interfering substances
  • Provide interpretive comments to help clinicians understand the significance of the results
  • For positive results, include antifungal susceptibility testing results to guide therapy

Key Takeaways

  • Diagnosis of deep and systemic mycoses requires careful specimen collection, processing, and analysis
  • Select the appropriate specimen type based on the suspected site of infection
  • Use aseptic technique and collect an adequate amount of material
  • Perform direct microscopic examination, culture, and other diagnostic tests to identify fungal pathogens
  • Report all results clearly and accurately, and provide interpretive comments to guide clinical decision-making

Key Terms

  • Dimorphic Fungi: Fungi that can exist in two different forms (e.g., mold and yeast) depending on environmental conditions (e.g., temperature). Examples include Histoplasma capsulatum, Blastomyces dermatitidis, and Coccidioides immitis
  • Opportunistic Fungi: Fungi that typically do not cause disease in healthy individuals but can cause severe infections in immunocompromised patients. Examples include Candida, Aspergillus, and Pneumocystis
  • Granuloma: A mass of immune cells (macrophages, lymphocytes) that forms in response to chronic inflammation or infection. Granulomas are often seen in fungal infections like histoplasmosis and coccidioidomycosis
  • Septate Hyphae: Hyphae that are divided into compartments by cross-walls (septa). Many common molds, such as Aspergillus and Fusarium, have septate hyphae
  • Non-Septate Hyphae: Hyphae that lack cross-walls (septa), forming a continuous, multinucleated cell. The Mucorales (Zygomycetes) fungi, such as Rhizopus and Mucor, have non-septate hyphae
  • Conidiophore: A specialized hyphal structure that produces conidia (asexual spores). The shape and arrangement of conidiophores are important characteristics for identifying molds
  • Arthroconidia: A type of asexual spore formed by the fragmentation of hyphae. Coccidioides immitis produces arthroconidia in its mold form
  • Spherule: A large, spherical structure containing endospores, formed by Coccidioides immitis in infected tissues
  • Germ Tube: A small outgrowth from a yeast cell that represents the beginning of hyphal formation. Germ tube formation is a characteristic used to identify Candida albicans
  • Pseudohyphae: Chains of elongated yeast cells that resemble hyphae but are formed by budding rather than true hyphal growth. Candida species often produce pseudohyphae
  • Macrophage: A type of immune cell that engulfs and digests foreign particles and pathogens. Macrophages play a key role in the host response to fungal infections
  • Galactomannan: A polysaccharide component of the cell wall of Aspergillus species. Galactomannan antigen detection is used to diagnose invasive aspergillosis
  • Beta-D-Glucan: A polysaccharide found in the cell walls of many fungi. Beta-D-glucan detection is used as a broad marker for invasive fungal infections
  • Voriconazole: A triazole antifungal drug commonly used to treat invasive fungal infections, particularly aspergillosis
  • Amphotericin B: A polyene antifungal drug used to treat a wide range of systemic fungal infections. It can be administered by IV
  • Echinocandins: A class of antifungal drugs that inhibit the synthesis of beta-glucan in fungal cell walls. Echinocandins are used to treat invasive Candida and Aspergillus infections