Blastomycosis is a systemic fungal infection caused by Blastomyces, a thermally dimorphic fungus. It exists as a mold in the environment but converts to a pathogenic yeast form within human tissue. The infection is endemic primarily to the midwestern, south-central, and southeastern United States, particularly the Ohio and Mississippi River Valleys and the Great Lakes region. Blastomycosis presents with varied symptoms, ranging from a mild, flu-like illness to severe respiratory distress or life-threatening disseminated disease involving the skin, bones, or central nervous system. Due to this varied presentation, a rapid and accurate diagnosis is necessary. Microscopic identification of the fungus in clinical samples serves as a fast initial step in the diagnostic process.
Obtaining the Sample for Microscopic Examination
The first step in identifying the infection is collecting a suitable clinical specimen based on the site of infection. For pulmonary cases, which are the most common presentation, non-invasive samples like sputum or invasive samples such as bronchoalveolar lavage (BAL) fluid are frequently used. Sputum is often collected first because it is easily expectorated and can yield fungal elements.
When the infection has spread beyond the lungs, other specimen types are required. These include scrapings or biopsies taken directly from skin lesions, the most common site of extrapulmonary dissemination. For deeper or localized disease, tissue biopsies from affected organs, aspirated pus from abscesses, or cerebrospinal fluid may be collected. The appropriate choice of specimen ensures that the laboratory technician has the best chance of finding the characteristic fungal structures for a presumptive diagnosis.
Rapid Microscopic Screening Techniques
Direct microscopic examination provides the fastest method for a presumptive diagnosis of blastomycosis. Specialized preparations enhance the visibility of fungal cells against the background of host tissue and cellular debris.
Potassium Hydroxide (KOH) Preparation
The Potassium Hydroxide (KOH) preparation is a quick and inexpensive technique. A strong alkaline solution, typically 10% KOH, is mixed with the specimen on a slide. This solution digests and clears away host cells, keratin, and other proteinaceous material. Since fungal cell walls are resistant to this digestion, Blastomyces yeast cells stand out clearly against the cleared background. This technique provides results within minutes.
Calcofluor White (CFW) Stain
The Calcofluor White (CFW) stain is often used alongside KOH. CFW is a fluorescent dye that binds specifically to the chitin and cellulose in fungal cell walls. When viewed under a fluorescence microscope using ultraviolet light, the fungal elements glow with a bright apple-green or blue-white color. This fluorescence makes even sparsely present fungal cells much easier to detect than with standard light microscopy, significantly increasing the sensitivity of the rapid screen.
Recognizing the Distinctive Fungal Morphology
Once the specimen is prepared, the technician searches for the distinctive morphology of the Blastomyces yeast form. The yeast cells are large, typically measuring 8 to 15 micrometers (µm) in diameter. They possess a thick, refractile cell wall that gives them a double-contoured appearance under the microscope.
The most characteristic feature is broad-based budding, where a daughter cell forms and remains attached to the mother cell by a wide connection. This broad connection is a key indicator for identification. When viewed in rapid mounts like KOH, these large, thick-walled, broad-based budding yeasts offer a strong presumptive diagnosis.
Histological Stains for Tissue Sections
In fixed tissue sections, fungal elements are less apparent with standard hematoxylin and eosin (H&E) staining, which is why special histological stains are employed. The Gomori Methenamine Silver (GMS) stain is commonly used, staining the fungal cell walls dark black or brown against a light green background. The Periodic Acid-Schiff (PAS) stain is also effective, highlighting the fungal cell walls in a bright magenta color. Both GMS and PAS stains help visualize the characteristic size and broad-based budding structure within the complex architecture of tissue.
Confirmatory Laboratory Testing
Although the distinctive morphology seen microscopically is typically considered a presumptive diagnosis, definitive confirmation requires subsequent laboratory testing. Fungal culture remains the gold standard because it allows for the isolation and growth of the organism from the clinical specimen.
The major limitation of culture is the slow growth rate of Blastomyces, which can take one to four weeks for positive identification. Once growth is detected, a commercially available DNA probe can quickly confirm the fungus’s identity. Because of the delay inherent in culture, non-microscopic methods are increasingly used to provide a faster and more comprehensive diagnosis.
Non-Microscopic Methods
Antigen detection, commonly performed on urine or serum samples using an enzyme immunoassay (EIA), offers a rapid, non-invasive method with high sensitivity. This test detects a fungal cell wall component shed into the body fluids of an infected patient. Molecular techniques such as Polymerase Chain Reaction (PCR) are also available in specialized reference laboratories to detect the fungus’s genetic material directly from clinical specimens. These complementary methods help validate the initial microscopic findings, ensuring an accurate diagnosis for appropriate treatment planning.