Systemic

This section covers systemic fungal infections and the crucial role of blood and bone marrow specimens in their diagnosis. Systemic mycoses represent a significant threat, particularly in immunocompromised individuals, and rapid, accurate detection is paramount. We’ll explore the specific techniques for collecting and analyzing these specimens, focusing on the unique challenges and diagnostic approaches for bloodborne and bone marrow-based fungal infections

Systemic Mycoses: A Critical Overview

• Nature of Infection: Systemic fungal infections involve the bloodstream and/or multiple organs, often leading to severe illness and even death
• High-Risk Patients: Immunocompromised individuals (e.g., transplant recipients, patients with HIV/AIDS, those undergoing chemotherapy) are particularly vulnerable
• Key Pathogens: Common culprits include Candida, Aspergillus, Cryptococcus, Histoplasma, and Coccidioides
• Diagnostic Imperative: Timely and accurate diagnosis is crucial for initiating appropriate antifungal therapy and improving patient outcomes

Specimen Types and Collection Techniques

Blood

• Purpose: To detect fungemia (fungi in the bloodstream), which indicates a disseminated infection
• Specimen Types
  • Whole Blood
    • Collection: Collected via venipuncture. Follow strict aseptic technique to avoid contamination
    • Volume: Collect the appropriate volume as specified by the blood culture system manufacturer. Underfilling bottles can lead to false negatives
    • Number of Sets: Collect multiple sets of blood cultures (typically 2-3) from different venipuncture sites to increase the sensitivity of detection. Collect over a short period of time (e.g., within an hour)
    • Timing: Collect blood cultures before administering antibiotics, if possible. If the patient is already on antibiotics, collect the cultures as close to the next dose as possible (when the antibiotic levels are likely to be lowest)
  • Lysed Blood
    • Collection: Whole blood is processed to lyse (break open) the blood cells, releasing any intracellular fungi
    • Purpose: To enhance the detection of fungi that may be sequestered within blood cells (e.g., Histoplasma)
  • Serum/Plasma
    • Collection: Whole blood is collected in tubes with or without anticoagulants, and the serum or plasma is separated by centrifugation
    • Purpose: Used for antigen and antibody detection assays

Bone Marrow

• Purpose: To diagnose disseminated fungal infections that involve the bone marrow
• Specimen Types
  • Bone Marrow Aspirate
    • Collection: A sample of liquid bone marrow is aspirated using a needle
    • Site: Typically collected from the posterior superior iliac crest (hip bone) or the sternum
    • Technique: Strict sterile technique is essential. The aspirate is usually collected into a syringe containing an anticoagulant (e.g., heparin)
    • Smears: Prepare smears of the bone marrow aspirate for microscopic examination
  • Bone Marrow Biopsy
    • Collection: A core of bone marrow tissue is removed using a special needle
    • Advantages: Provides a more representative sample of the bone marrow architecture than aspirate alone
    • Processing: The biopsy specimen is fixed, sectioned, and stained for microscopic examination
    • Combined Approach: Bone marrow aspirate and biopsy are often performed together to provide a comprehensive assessment

General Considerations for Systemic Specimens

• Aseptic Technique: Meticulous aseptic technique is paramount during collection to prevent contamination, which can lead to false-positive results
• Volume: Collect the appropriate volume of blood or bone marrow as specified by the laboratory protocol
• Timely Processing: Transport specimens to the laboratory promptly. Delays can affect the viability of fungi and the accuracy of test results
• Communication: Inform the laboratory about the patient’s clinical history, suspected fungal pathogen, and any relevant medications (e.g., antibiotics, antifungals)
• Safety: Handle specimens with appropriate precautions, as some systemic fungi can pose a risk to laboratory personnel

Laboratory Analysis

Blood

• Blood Culture
  • Automated Systems: Most clinical laboratories use automated blood culture systems that continuously monitor the blood culture bottles for signs of microbial growth (e.g., changes in CO2 levels, pH)
  • Incubation: Blood culture bottles are incubated for up to 5-7 days, or longer if slow-growing fungi are suspected
  • Gram Stain: When a blood culture bottle flags positive, a Gram stain is performed to determine if bacteria or fungi are present
  • Fungal Identification: If fungi are detected, the isolate is identified using a variety of methods (e.g., colony morphology, microscopic examination, biochemical tests, MALDI-TOF MS, molecular methods)
• Lysed Blood Culture
  • Lysis-Centrifugation: A technique used to concentrate fungi from lysed blood
  • Microscopic Examination: The sediment is examined microscopically for fungal elements
  • Culture: The sediment is also cultured on appropriate fungal media
• Antigen Detection
  • Galactomannan Assay: Detects galactomannan, a polysaccharide component of the cell wall of Aspergillus species. Used to diagnose invasive aspergillosis
  • Beta-D-Glucan Assay: Detects beta-D-glucan, a component of the cell wall of many fungi. Used as a broad marker for invasive fungal infections
  • Cryptococcal Antigen Assay: Detects Cryptococcus capsular polysaccharide antigen in serum or CSF. Used to diagnose cryptococcosis
  • Histoplasma Antigen Assay: Detects Histoplasma antigen in serum or urine. Used to diagnose histoplasmosis
• Molecular Detection (PCR)
  • Detects fungal DNA in blood samples
  • Highly sensitive and specific
  • Can be used to identify fungi directly from blood, even when blood cultures are negative
• Antibody Detection * Detects antibodies against specific fungi in serum * Examples: Coccidioides antibodies, Aspergillus antibodies

Bone Marrow

• Microscopic Examination
  • Smears: Bone marrow aspirate smears are stained with Wright-Giemsa stain and examined microscopically for fungal elements (e.g., Histoplasma within macrophages)
  • Touch Preparations: Imprints of the bone marrow biopsy specimen can be made on glass slides, stained, and examined microscopically
• Histopathology
  • Bone marrow biopsy specimens are fixed, sectioned, and stained with special stains (e.g., GMS, PAS) to highlight fungal organisms
• Culture
  • Bone marrow aspirate and biopsy specimens are cultured on appropriate fungal media
  • Incubate cultures for up to 4 weeks
• Molecular Detection (PCR)
  • Detects fungal DNA in bone marrow samples
  • Highly sensitive and specific
  • Can be used to identify fungi directly from bone marrow, even when cultures are negative

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 systemic mycoses requires careful specimen collection, processing, and analysis
• Blood and bone marrow specimens are essential for detecting disseminated fungal infections
• Use aseptic technique and collect the appropriate volume of specimen
• Perform blood cultures, antigen detection assays, and molecular tests on blood samples
• Examine bone marrow aspirate and biopsy specimens microscopically, culture them, and perform molecular tests
• Report all results clearly and accurately, and provide interpretive comments to guide clinical decision-making

Key Terms

• Fungemia: The presence of fungi in the bloodstream
• Disseminated Infection: An infection that has spread from a localized site to multiple organs or body systems
• Neutropenia: A condition characterized by a low number of neutrophils (a type of white blood cell) in the blood. Neutropenic patients are at increased risk for invasive fungal infections
• Central Venous Catheter (CVC): A catheter inserted into a large vein (e.g., subclavian, internal jugular) to administer medications, fluids, or nutrition. CVCs can be a source of bloodstream infections, including candidemia
• Biofilm: A community of microorganisms (e.g., bacteria, fungi) that adhere to a surface and are encased in a matrix of extracellular polymeric substances. Biofilms can form on medical devices (e.g., catheters) and make infections more difficult to treat
• Minimum Inhibitory Concentration (MIC): The lowest concentration of an antifungal drug that inhibits the visible growth of a fungus in vitro. MIC values are used to determine antifungal susceptibility
• Etest: A commercially available method for determining antifungal MIC values. Etest strips contain a gradient of antifungal concentrations and are placed on agar plates inoculated with the test organism
• AFST: Antifungal Susceptibility Testing
• Mould-Specific Identification: Refers to laboratory techniques, including microscopic examination and molecular assays, used to accurately identify specific mold species in clinical samples, aiding in diagnosis and treatment of infections caused by these fungi
• Panfungal PCR: is a molecular diagnostic technique that utilizes polymerase chain reaction (PCR) to amplify and detect conserved DNA sequences present in a wide range of fungi, allowing for the identification of fungal infections, even when traditional culture methods are negative or impractical