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Case twenty nine: the synergistic role of microbiology and pathology.

Case Follow up

Serologic tests for antibodies to Histoplasma and Blastomyces by immunodiffusion were negative. The urine Histoplasma antigen test was positive on two separate occasions. The histoplasma antigen test may be falsely positive with an infection from Blastomyces dermatiditis because the antibodies used in the reagent cross-reacts with antigens from both Histoplasma and Blastomyces. Our case demonstrates that ancillary studies don't have the characteristic high sensitivity or specificity of fungal cultures and should be interpreted with caution.

Laboratory Specimen Processing and Analysis

Two shave biopsies of the nodules involving the nasal tip and anterior thigh revealed similar findings. The epidermis is irregular and thickened with few collections of neutrophils (Figure 2A). The dermis revealed a diffuse and focally nodular mixed infiltrate of lymphocytes, neutrophils, and small non-caseating granulomas, some of which contained yeast (Figure 2B and 2C). A single yeast revealed definitive broad-based budding diagnostic of blastomycosis (Periodic acid-Schiff with Diastase, Figure 2D).

A fragment of tissue was sent for fungal culture. On Sabouraud Dextrose Agar at 30[degrees] C., a fluffy white homogeneous colony appeared after incubation (Figure 3A). Since conidia were not seen in this colony, the organism was subcultured to Potato-flake agar at 30[degrees] C. The mold conidiated, and the lactophenol cotton blue preparation showed classic "lollipop sticks", or short, single, unbranched conidiophores with round-to-oval conidia (Figure 3B). The mold was then subcultured to Brain Heart Infusion agar and incubated at 37[degrees] C, which yielded yeast colonies. The wet mount showed yeast with broad-based buds. These characteristics are diagnostic for Blastomyces dermatitidis.

Introduction

Blastomyces dermatitidis is a dimorphic fungus that is found in the soil of the Midwestern states, and transmission of the mold is linked to gardening and outdoor recreational activities. Pulmonary blastomycosis followed by cutaneous blastomycosis are the most common acute and chronic presentations, respectively. Cutaneous blastomycosis may be seen without coexistent pulmonary symptoms. Usually cutaneous blastomycosis is a secondary event and follows hematogenous seeding of the yeast form from the lung during a time of stress. Cutaneous blastomycosis may occur in both normal and immunocompromised patients. However, patients with suppressed cell-mediated immunity have a greater number of organisms seen in tissue. (1) Immunosuppressed patients are also at a higher risk of developing a more severe systemic disease. (2,3)

Epidemiology

Blastomyces dermatitidis is a dimorphic fungus that is found in the soil of the Ohio River valley and regions surrounding the Mississippi river within the US. Soil in these regions provide an acidic environment with abundant organic compounds that provides the right environment for molds to grow. (5,6) Infections may also occur in regions surrounding the great lakes such as Ontario and the Midwest, as well as Southeastern states. (7,8) Rare reported cases have been found in Africa, South America, and Europe. (9,10) Blastomycosis is more prevalent in men which is correlated with the fact that men more frequently hold outdoor occupations such as lumberjacks, fishermen, and construction workers. (11) However, gardeners and outdoor recreational activities also are known modes of exposure. Transmission of the conidia from the molds in soil via aerosol inhalation is the most common method of introduction of the organism to the host. Blastomycosis is not contagious and does not require isolation of the affected individual.

Pathophysiology

A wide spectrum of clinical manifestations may occur with blastomycosis. Infections can range from asymptomatic to disseminated disease. Transient self-limited pneumonia is the most common presentation and may be asymptomatic or manifest as nonspecific influenza-like symptoms of low grade fevers, prodromal symptoms, and nonproductive cough. Lung infections comprise 60-75% of all cases of blastomycosis. (12) Generally, symptoms persist for 2 to 12 weeks. Disseminated blastomycosis occurs weeks to months later when cell-mediated immunity is unable to clear up the fungal organisms. The thick wall of Blastomyces dermatitidis make these organisms harder to clear for macrophages. Spores convert to the yeast form in the lungs at 37[degrees] C. The yeast can then disseminate via the blood stream or lymphatics.

Disseminated or systemic blastomycosis also has a spectrum of manifestations. The commonest site (70%) of disseminated blastomycosis is the skin. Even in the absence of a prior documented pulmonary infection, cutaneous blastomycosis is assumed to be secondary to dissemination from the lung, unless a documentable direct skin exposure or trauma is present. (13,14) Cutaneous lesions include boils, abscesses, and verrucous plaques with peripheral pustules, with or without ulcerations, overlying any of the aforementioned lesions. Common skin sites include the face and arms. Other common sites of infection include genitourinary tract, and central nervous system. (1,15,16)

Specimen Collection

The type of specimen required depends on the clinical presentation and diagnostic modality. For pulmonary involvement, cultures of bronchiolar lavage fluid are more sensitive than sputum. The advancing border of cutaneous lesions is more likely to yield a diagnosis than central necrotic tissue, aspirates, pus, or fine needle aspirates of fluid material. When possible, collection of a generous tissue biopsy is recommended to increase sensitivity. For pathology, the specimen should be placed immediately in formalin for fixation. For fungal cultures, the specimen should be transported to the microbiology lab at room temperature within two hours of collection. Blastomyces dermatitidis requires oxygen for growth; therefore, anaerobic transport media should never be used. A test for Blastomyces antigen in serum, urine, or CSF is available and may be helpful in making a diagnosis. (27)

Pathology Interpretation

Well developed skin lesions will show pseudo-epitheliomatous hyperplasia, intraepidermal neutrophilic microabscesses, and a dense suppurative and granulomatous dermal infiltrate with giant cells. Pseudoepitheliomatous hyperplasia may mimic squamous cell carcinoma. Single large yeasts measuring 8-20 [micro]m are found within histiocytes, giant cells, abscesses, or extracellularly. (18,19) Broad-based budding yeasts, when present, are virtually diagnostic of Blastomyces dermatitidis. Yeast numbers are most abundant in late, well-developed lesions. In early lesions, all of the characteristic histological features will not be present and diagnostic broad-based budding yeasts are difficult to find. For such cases, ancillary special stains for fungus should be deployed. These stains highlight the cell walls and make recognition of budding yeast less difficult.

Laboratory Medicine in Microbiology

At our institution, we use Sabouraud Dextrose, Brain Heart Infusion with 10% sheep blood with and without Chloramphenicol and Gentamicin, Inhibitory Mold Agar (Chloramphenicol), and Mycosel (Chloramphenicol and Cycloheximide). Sabouraud Dextrose is a standard non-enriched media which provides nutrients suitable to 75% of fungi and some bacterial contaminants. (26) Adding Inhibitory Mold Agar increases the sensitivity of fungal cultures by 25%, hence at the minimum a Sabouraud Dextrose and Inhibitory Mold Agar are used by many labs. (26) Brain Heart Infusion is an enriched media which provides additional nutrients necessary for certain fungi. Chloramphenicol and gentamicin are antibiotics that together suppress the majority of gram negative and gram positive bacteria thus facilitating fungal growth in cases where bacteria are present in the specimen. Mycosel is a highly selective media, which contains cycloheximide to suppress the growth of many fungi; however, the agents of endemic mycosis, dermatophytes, and Candida albicans are resistant to cycloheximide and will grow. (27)

The media are incubated at 25-30[degrees] C for 4 weeks with routine monitoring for growth. Blastomyces dermatitidis produces beige to white fluffy colonies in 5-10 days, often with folds. (28) Potato glucose agar and potato flake agar will show 0.5-3 cm colonies after 7 days. (27) Sealed media plates are transferred to a hood where they can be opened and a wet mount can be prepared. Upon identifying the "lollipop stick" morphology, one's suspicion towards a possible Blastomyces dermatitidis should rise, prompting a confirmatory test. These include subculture of the mold to a Brain Heart Infusion at 37[degrees]C, or use of the AccuProbe Hybridization test. Within 7-14 days, the mold should convert to yeast phase and give small verrucous, white-beige creamy colonies. (27) A lactophenol blue preparation from the yeast colonies will reveal 5-15 [micro]m large, broad-based budding yeast. (24,27)

Hence, the gold standard of diagnosis is a combination of dermatopathology and microbiology. The dermatopathology can definitively show morphologic features of the skin's response to the infection and perhaps yeast. The microbiology has a much higher sensitivity and will confirm presence of the organism in tissue.

Ancillary Tests and Confirmation

Two common ancillary tests used in the confirmation of blastomycosis are Blastomyces antigen and Immunodiffusion assay tests. The antigen test can be done on urine, serum, or CSF and utilizes sandwich ELISA technology. The urine antigen has 90-93% sensitivity; however, the specificity is limited as the antibodies employed in this assay show considerable cross reactivity with antigens of Paracoccidiodes & Histoplasma. (24,27) Conversely, the Histoplasma antigen test shows similar cross-reactivity and may be falsely positive in cases of Blastomycosis. The immunodiffusion assay is a double microimmunodiffusion test in which Blastomyces antigen A (or less commonly antigen WI-1) is applied to a center well within the gel. The patient's serum is applied to peripheral wells along with a positive control. The presence of a Blastomyces antibody within the patient's sera is detected if precipitin lines form. This test shows 30% sensitivity for localized infections and 90% for disseminated infections. (27) As is evident, due to the lack of specificity by the antigen test and sensitivity by the antibody test, these methods are better reserved for confirmation.

Conclusion

As seen in our case, cutaneous blastomycosis can be difficult to diagnose given its nonspecific presentation. Serologic test results can be delayed since they are often send outs. Furthermore, these tests are not specific, limiting their utility during a first diagnosis. Blastomyces and Histoplasma antigen tests, both show cross reactivity for the other organism. Under such circumstances, the synergistic use of tissue diagnostic techniques and microbiology is paramount to render an accurate diagnosis.

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1. Blastomyces dermatitidis is best characterized as:

A. Dimorphic fungi with narrow based budding and thick PAS positive capsule.

B. Dimorphic fungi with small round yeasts 2-4 pm found within histiocytes.

C. Dimorphic fungi with high prevalence in Southwestern states.

D. Dimorphic fungi with large broad based buds 10-15 pm.

2. The purpose of subculturing fungi from Sabouraud Dextrose at 25-30[degrees]C onto the Brain Heart Infusion at 37[degrees]C is to convert:

A. Large molds into smaller molds for definitive identification.

B. Small yeasts into larger molds for definitive identification.

C. Molds into yeasts for definitive identification.

D. Yeasts into molds for definitive identification.

3. Chloramphenicol and Gentamicin are often found in fungal cultures because:

A. These antibiotics suppress the growth of competing gram negative bacteria.

B. These nutrients promote the growth of dimorphic fungi.

C. These antibiotics suppress the growth of gram positive bacteria.

D. These nutrients promote sporulation by molds.

4. Blastomycosis is often seen in individuals who:

A. Take long hot baths in bathtubs.

B. Enjoy sand based outdoor activities in the deserts of Arizona.

C. Hike hills along the Mississippi river without protective clothing.

D. Breed cats and dogs for a living.

5. The preparation of Blastomyces dermatitidis mold for visualization by lactophenol cotton blue yields the classic morphology of:

A. "Birds on a wire".

B. "Lollipop" conidiophores.

C. "Mariner's wheel" yeast.

D. "Snowshoe" macroconidia.

6. The gold standard for diagnosis of Blastomycosis is:

A. Tissue biopsy diagnosis.

B. Fungal culture with tissue biopsy diagnosis.

C. Immunodiffusion assay for Blastomyces antibody.

D. Sandwich ELISA for Blastomyces antigen.

7. When collecting tissue sample for either histology or cultures, the most appropriate sample is:

A. Pus or necrotic debri from the center of an abscess.

B. Fine needle aspirate of fluid material.

C. Minute amounts of tissue at the advancing border of the skin lesion.

D. Large amounts of tissue at the advancing border of the skin lesion.

8. Cutaneous blastomycosis is widely accepted to occur usually by which route:

A. Direct inoculation of mold contaminated soil

B. Deep skin puncture by cat or dog bite or scratches with yeast inhabitant

C. Wide dissemination of the skin by the way of lymphatic system.

D. Hematogenous spread weeks to months after a healed pulmonary infection.

9. Scognamiglio et al. stated that many laboratories currently use which initial media regimen to increase the sensitivity of fungal cultures to close to hundred percent.

A. Mycosel

B. Sabouraud Dextrose & Mycosel

C. Sabouraud Dextrose & Inhibitory Mold Agar

D. Sabouraud Dextrose & Brain Heart Infusion

10. Which clinical manifestation best describes cutaneous blastomycosis:

A. Numerous erythematous nodules and abscesses of the skin.

B. Flat red and white stippled macules and papules

C. Black colored plaques and patches with ulcerated centers

D. Sloughing warty protuberances of the skin.

EDITOR'S NOTE: BEFORE reading the Case Follow-up and Discussion below, study the Case Description on page 82 of this issue, and formulate your own answers to the questions posed.

References

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(8.) Klein BS, Vergeront JM, Davies JP. Epidemiological aspects of blastomycosis, the enigmatic systemic mycosis. Semin Respir Infect. 1986;1:29-39.

(9.) Centers for Disease Control and Prevention: Blastomycosis acquired occupationally during prairie dog relocation-Colorado, 1998. JAMA. 1999;282(1):21-22.

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(11.) Vasquez JE, Mehta JB, Agrawal R, Sarubbi FA. Blastomycosis in northeast Tennessee. Chest. 1998;114(2):436-443.

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(15.) Eikenburg HU, Amin HU, Lich R. Blastomycosis of the genitourinary tract. J Urol. 1975;113:650-652.

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(17.) J Michael D., M Scott T., H Courtney P. Cutaneous Blastomycosis Mimicking Squamous Cell Carcinoma of the Scrotum. The Internet Journal of Urology. 2007;5(2).

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(19.) Rippon JW, 1974. Medical Mycology: The Pathogenic Fungi and the Pathogenic Actinomycetes. Philadelphia, Pa: WB Saunders, 297-320.

(20.) Hussain Z, Martin A, Youngberg G. Blastomyces dermatitidis with Large Yeast Forms. Arch Pathol Lab Med. 2001;125: 663-664.

(21.) Ware AJ, Cockerell CJ, Skiest DJ, Kussman HM. Disseminated sporotrichosis with extensive cutaneous involvement in a patient with AIDS. J Am Acad Dermatol. 1999;40(2):350-355.

(22.) Von Lichtenberf F, 1991. Pathology of Infectious Diseases. New York: Raven Press, 201-248.

(23.) Padhye AA, Hampton AA, Hampton MT, Hutton NW, Prevost-Smith E, Davis MS. Chromoblastomycosis Caused by Exophilia spinifera. Clinical Infectious Diseases. 1996;22:331-335.

(24.) Barnhill R, 2010. Dermatopathology. China: McGraw-Hill, 468-476.

(25.) Rajan J, Mathai AT, Prasad P, Kaviarasan P K. Multifocal Tuberculosis Verrucosa Cutis. Indian J Dermatol. 2011 May-Jun; 56(3): 332-334.

(26.) Scognamiglio T, Zinchuk R, Gumpeni P, Larone DH. Comparison of Inhibitory Mold Agar to Sabouraud Dextrose Agar as a Primary Medium for Isolation of Fungi. J Clin Microbiol. 2010 May; 48(5): 1924-1925.

(27.) Versalovic J, 2011. Manual of Clinical Microbiology. District of Columbia: ASM Press, 1902-1942.

(28.) Reiss E, Shadomy H, Lyon G, 2012. Fundamental Medical Mycology. New Jersey: Wiley-Blackwell, 125-138.

Ravi Patel, Judith Rhodes, Joel Mortensen, Dept. of Pathology and Laboratory Medicine, University of Cincinnati Medical Center (Judith Rhodes also Clinical Laboratories, UC Health; Joel Mortensen also Dept. of Pathology and Laboratory Medicine, Cincinnati Children's Medical Center); Kerith E. Spicknall, Dept. of Dermatology, University of Cincinnati
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Title Annotation:CASES IN CLINICAL MICROBIOLOGY
Author:Patel, Ravi; Rhodes, Judith; Spicknall, Kerith E.; Mortensen, Joel
Publication:Journal of Continuing Education Topics & Issues
Article Type:Clinical report
Date:Aug 1, 2014
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