Answers and Discussion

 

Final diagnosis and Discussion of Differentials

In this case, the H&E-stained section of the skin lesion shows a few spherical thick-walled spores admixed with neutrophils, histiocytes, and multinucleated foreign body giant cells (pyogranulomatous inflammation) (Figure 1). By GMS stain, the spores show characteristic broad-based budding (Figure 2). The organisms grow on Sabouraud dextrose agar at room temperature (Figure 3) and show septate hyphae with short conidiophores and lollipop-like microconidia evidenced by lactophenol aniline blue stain (Figure 4). Therefore, the characteristic morphological features support the organism identified in this case was Blastomyces dermatitidis (B. dermatitidis).

Blastomyces spp. are thermally dimorphic fungi. In addition to B. dermatitidis, DNA studies demonstrated at least a second distinct species within the genus Blastomyces, B. gilcristii. Both species demonstrate identical growth characteristics and morphology. Blastomyces spp. grow as molds at room temperature and the mold form may convert to yeast with incubation at 37°C. Although smaller yeast forms (microforms) have been reported, the yeast cells of Blastomyces spp. are usually 8-15 µm in diameter and spherical with single broad-based budding, multibudding occasionally is seen. They are naturally found in soil and decomposing wood and leaves. People get infected by inhaling airborne conidiospores that produce initial infection in the lungs, and infection can also be acquired by direct cutaneous inoculation. Dogs can become infected with Blastomyces spp. In the United States, blastomycosis is endemic in the areas of the Ohio River and Mississippi River, Great Lakes, and St. Lawrence riverway. It commonly affects people who often work outdoors or who engage in outdoor recreational activities like hunting. The clinical presentations are variable from asymptomatic infection to fatal disseminated disease with multiple organs or systems involvement including lungs, skin, bone, and central nervous system. Skin is one of the most common sites involved. Cutaneous blastomycosis (also known as Gilcrest’s Disease) typically presents as ulcerative and/or verrucous lesions, grossly resembling squamous cell carcinoma, basal cell carcinoma or pyoderma gangrenosum, particularly when micro-abscesses coexist.

Laboratory tests are essential in diagnosing blastomycosis. The mold morphology on lactophenol aniline blue preparations is not unique to Blastomyces spp. and can be seen in several genera of molds, hence, conversion to the yeast form or evaluation by molecular studies is necessary for definitive diagnosis. Yeast conversion is not commonly performed currently due to the several week timeframes required so molecular techniques are favored. In this case, conversion was unnecessary in that the typical yeast form existed in the tissue biopsy. Fresh wet preparations of clinical specimens can be applied to directly visualize organisms with addition of potassium hydroxide (KOH) to increase visibility of the organisms. As is the case with other fungi, Calcofluor White causes Blastomyces spp. to fluoresce and may be applied directly to KOH preparations. An antigen assay that detects a galactomannan component in the cell wall of Blastomyces can be used to test urine, serum, BAL fluid, and CSF specimens but may be false-positive with histoplasmosis and certain other fungal infections. B. dermatitidis are positive for GMS stain, Congo red stain, and Periodic acid-Schiff (PAS) stain and negative for Gram stain. PCR-based tests show a good specificity however their clinical application is not yet confirmed.

 Histoplasma capsulatum

Clinically, histoplasmosis presents similarly as blastomycosis. Like blastomycosis, histoplasmosis is endemic in Mississippi and Ohio River valleys and acquired via inhalation of fungal microconidia and the infected patients can present with skin lesions. Histoplasma spp. are also dimorphic fungi and found in soil or caves containing bird or bat guano. In contrast to B. dermatitidis, the yeasts of Histoplasma spp. are smaller, oval shaped with narrow-based budding. The Congo red stain can be performed to differentiate the two types of yeasts, for which H. capsulatum is usually negative.

Additionally, the yeast cells of Histoplasma tend to cluster and present as phagocytized organisms in macrophages and monocytes although extracellular clusters can also be seen. Visualization of those characteristic features are also helpful to make a definitive diagnosis of histoplasmosis.  

Coccidioides immitis

Coccidioides spp. (C. immitis and C. posadasii) are dimorphic, though not thermodimorphic, fungi that exhibit large, thick-walled spherules containing numerous endospores in tissue with growth as molds on synthetic mycology media. While the tissue form is usually that of spherules, occasionally hyphae with arthroconidia can also be detected in tissue. Microscopically, Coccidioides spp. can potentially simulate Blastomyces spp. when free endospores are found outside of spherules and two endospores abut one another, suggesting broad-based budding of yeast.

Coccidioides spp. are found in soil in southwestern United States and parts of Mexico and Central and South America. Infection is acquired by inhalation of aerosolized fungal arthroconidia. Coccidioidomycosis presents primarily as self-limiting pulmonary disease and less commonly leads to a disseminated disease. Cutaneous manifestations have been reported presenting as exanthema in response to acute pulmonary or to a disseminated infection, or as a primary infection from direct inoculation of the organisms.

Cryptococcus spp.

Cryptococcus spp. are spherical to oval yeast cells with polysaccharide capsules. Compared to Blastomyces spp., which are larger (8-15 µm in diameter) and with broad-based budding, Cryptococcus spp. are usually smaller in size (3-10 µm in diameter) and with narrow-based budding. Microscopically, nonbudding cells of B. dermatitidis can be mistaken as Cryptococcus spp., which can be differentiated by special stains. Cryptococcus neoformans/gattii are positive for capsular stains like mucicarmine and alcian blue. Sometimes less capsule is produced so-called “capsular deficient” and these may demonstrate less capsular staining.  Cryptococcus neoformans/gattii are positive for Fontana-Masson stains, while for which B. dermatitidis would be negative.  For Cryptococcus spp. with capsules, the yeast cells show stronger positivity for mucicarmine stain than B. dermatitidis which may produce only slight or negative staining.

Cryptococcus neoformans (C. neoformans) and Cryptococcus gattii (C. gattii) are two main pathogenic species. Cryptococcus spp. infections occur in people who have weakened immune systems, particularly those who have advanced HIV/AIDS. Cryptococcus spp. are found in soils in roosting sites of pigeons or areas frequented by pigeons and chickens. People become infected after inhaling the yeasts. Infections caused by C. gattii may more often occur in immunocompetent individuals than those caused by C. neoformans. In general, cryptococcosis present as pulmonary, CNS or skin disease. Clinical presentations vary based on individual’s immunocompetency.

References

1. Elena Gonzalez Caldito,Camila Antia, Vesna Petronic-Rosic. Cutaneous Blastomycosis. JAMA Dermatol. 2022;158(9):1064
2. Kathleen A. Linder, Carol A. Kauffman, and Marisa H. Miceli. Blastomycosis: A Review of Mycological and Clinical Aspects. J Fungi (Basel).2023 Jan; 9(1): 117.
3. Azar MM et al: Laboratory diagnostics for histoplasmosis. J Clin Microbiol. 55(6):1612-20, 2017
4. Gordon L. Love, Julie A. Ribes. Color Atlas of Mycology, 2018.
5. Sandra Cecilia Garcia Garcia, Julio Cesar Salas Alanis, Minerva Gomez Flores, Sergio Eduardo Gonzalez Gonzalez, Lucio Vera Cabrera, and Jorge Ocampo Candiani. Coccidioidomycosis and the skin: a comprehensive review. An Bras Dermatol.2015 Sep-Oct; 90(5): 610–619.
6. ExpertPath (https://app.expertpath.com/main)
7. CDC (https://www.cdc.gov/fungal/index.html)

Board type review questions

1.  A middle-aged man from Mississippi presented to the emergency department with fever, cough, myalgia, and increased fatigue. The patient has a positive history of HIV infection and is on anti-retroviral therapy. Chest x-ray revealed enlargement of bilateral hilar lymph nodes. Fine needle aspiration of lymph nodes was performed, and the specimen was submitted for cytologic evaluation. Diff-Quik stained cytospin shows numerous small yeast clusters within large macrophages. GMS stain reveals the yeasts are oval shaped with narrow-based budding. Which one of the following is the most likely causative agent of the clinical presentations?

1. Blastomyces dermatitidis
   
   
2. Histoplasma capsulatum
   
   
3. Cryptococcus spp.
   
   
4. Pneumocystis jiroveci

2. Which one of the following statements is true regarding blastomycosis?

1. Most blastomycosis infections are caused by inhalation of Blastomyces dermatitidis conidia.
   
   
2. Microscopic exams show spherical multinucleated yeast cells with single narrow-based budding and thick refractile wall.
   
   
3. The thick cell wall of blastomycosis inhibits a pyogranulomatous reaction.
   
   
4. Blastomyces dermatitidis grows on Sabouraud dextrose agar at 24°C, but not at 37 ˚C.