Sunday, 2 December 2012

Blastomyces dermatitidis

Blastomyces dermatitidis (Fungus) -Teleomorph = Ajellomyces dermatitidis

Ecology & More:  Blastomyces dermatitidis is a dimorphic fungus meaning its morphology can express two different forms primarily depending on temperature.  At ambient environmental temperatures (~25 to 30oC), Blastomyces is found in a filamentous mould form.  At body temperature (37oC) it grows in a discrete yeast form.   Blastomyces dermatitidis is most likely a soil saprobe (lives on decaying organic matter).   It is endemic in several locations of North America including eastern to central areas of Canada (Ontario-Manitoba border), the United States, particularly the Mississippi and Missouri valleys and can extend into Central America.  The isolate which appears below was isolated from the bronchial washes taken from a cottage owner in the Parry Sound area of Ontario.  Isolates have also been recovered from parts of the Middle East & Africa however reports suggest that they are serologically distinct showing geographic diversity.

Blastomyces dermatitidis is infrequently isolated directly from the environment, however acidic soils with rich organic debris such as decaying vegetative matter, animal excreta (providing increased nitrogen content), which is protected from direct sunlight may offer the optimal opportunity.

Pathogenicity:  Infection most likely occurs through inhalation of fungal spores when the environmental fungus is disturbed (raking leaves, moving woodpiles, gardening/planting, etc.).  Once inside the lungs, the change of environment (carbon dioxide content, organic nutrients, pH and primarily the increased temperature (37oC)), and causes the fungal spores to develop into the yeast cell form.  The yeast form is efficient in disseminating to other areas of the body.  Symptoms may not appear for up to 120 days post infection.   This organism is the etiologic agent of blastomycosis which may produce illness from a transient pulmonary infection to a chronic infection characterized by suppurative and granulomatous lesions in any part of the body.  While inhalation is the most common mode of infection, the fungus spores are thought to be able to gain entry through cuts and scrapes as well.  Infection can spread to skin and bones and other organs may also become involved.  Animals, such as dogs, can also acquire blastomycosis however there is no evidence that the fungus (in its yeast form) can be spread from animals nor from person to person. 

Early symptoms can mimic other pulmonary diseases from pneumonia, tuberculosis or even pulmonary carcinoma (lung cancer).  The patient may initially present with chest pains, night sweats and weight loss. Misdiagnosis delays therapy as antibacterials have no effect on fungal growth.  The prolonged therapy with the antifungal itraconazole has been successfully used to treat blastomycosis however it is the physician’s responsibility to determine the appropriate agent for treatment.  Yearly in Ontario, cases of blastomycosis usually occur in the low double digits.  As a result, physicians are usually not aware of this illness.  Misdiagnosis and inappropriate or delayed therapy can easily occur.  Sadly, correct diagnosis is often made at autopsy.

The isolate shown below was isolated from a brocheal alveolar lavage (BAL) obtained from a cottage owner in the Parry Sound (mid-northern) region of Ontario.  KOH & calcofluor white preparations were the first preparations examined under a UV microscope where fluorescent yeast-like cells showed tell-tale broad-based budding, characteristic of Blastomyces dermatitidis.  Unfortunately, our UV microscope no longer supports a camera mount and therefore no photographs were taken of this preparation.
A gram stained smear of the BAL specimen showed the same broad-based budding as seen below;

Blastomyces dermatitidis -gram stain of BAL showing broad-based budding yeast, characteristic of Blastomyces dermatitidis.  (X1000, Nikon)

*Keep in mind the dimorphic morphology of this fungus in the descriptions below.

Macroscopic Morphology

Grown at 25 to 30oC:
  •          Filamentous fungus form

  •          Moderately slow growth, usually maturing in about 2 weeks but suspect cultures should be held for 8 weeks prior to discarding as negative.

  •          Exhibits a cottony or downy texture.

  •         Colonies produce white areal hyphae on the surface which may turn a yellowish to tan colour as the colony ages.   The reverse is typically a light tan to brown.
Blastomyces dermatitidis - SAB, 7 days at 30oC

Grown at 37oC:
  •        Yeast form

  •        Slow to moderate growth

  •          Best chance for conversion is to mimic human conditions.  Fungus on enriched media (Blood Agar or Brain-Heart Infusion (BHI)) incubated in about 6% CO2. 

  •          Yeast form has been described as creamy, heaped or wrinkled, granular to verrucose (with projections).  The yeast form is cream to tan in colour.

  •          The yeast form of Blastomyces dermatitidis is inhibited by cycloheximide.
While the filamentous form of Blastomyces dermatitidis is the infectious stage of this dimorphic fungus, for safety's sake, no attempt was made to convert to the yeast phase.  To do so, one would inoculate fungal spores onto some rich media (blood agar, brain-heart infusion or even inoculate it into a blood culture bottle) and incubate in CO2b (~6-8%) to maximize conversion.  For that reason, no photographs of Blastomyces dermatitidis in yeast form are presented here.

Microscopic Morphology:
Grown at 25 to 30oC:

  •          Blastomyces dermatitidis produces septate hyphae

  •          Unbranched conidiophores of rather short, yet varying length extend from the hyphae

  •          Conidia are hyaline (clear) and are produced singly at the apex of the conidiophore or can develop directly on the hyphae.

  •          Conidia are unicellular, round to pyriform (tear-drop) in shape (~2 to 10 µm dia.)  Conidia at the terminal end of the conidiophore resemble a ‘lollipop’ in structure.
 Blastomyces dermatitidis - a low power view of hyphae growing away from the point of inoculation of a slide culture: ~48hrs, 30oC (100X, DMD-108)

Blastomyces dermatitidis - Conidia seen growing along hyphae. (400X, DMD-108)

Blastomyces dermatitidis - another view of the "lollipop-like" conidiophore-conidia structures extending along the length of hyphae.  (400X, DMD-108)

Blastomyces dermatitidis -  unbranched conidiophores are rather short, though of varying length and bear round to slightly pyriform (tear-drop) shaped conidia at the apex.
(1000X, DMD-108)

Blastomyces dermatitidis - ditto
(1000X, DMD-108)

Blastomyces dermatitidis - more is better, another photo as the above two.
(1000+10X, DMD-108)

Blastomyces dermatitidis - side by side comparison of the same photo taken through two different planes of focus.  The depth of field at this magnification doesn't capture the three dimensional nature of this fungus.  As the conidiophores and conidia project away from the hyphae, the focus is lost.  Septations in the hyphae are visible in the photo on the left.  The length of the conidiophores is not evident in these photos as the conidia may be growing right on the hyphae or the conidiophores may be projecting directly upwards towards the lens, masking their length.  The hyphae are in focus on the left, the conida more so on the right.
(1000+10X, DMD-108)

Blastomyces dermatitidis - right in the thick of things.
(1000+10X, DMD-108)

Blastomyces dermatitidis - and another at high magnification.
(LPCB 1000X, DMD-108)

Blastomyces dermatitidis - the last one.
(LPCB 1000X, DMD-108)

Once again, no attempt was made to convert the filamentous form to the yeast form so no photographs are presented below.
Grown at 37oC: (enhanced by rich media and CO2, or as may be found in infected tissue)

  •          Yeast-like cells (~8 to 15 µm dia.) exhibit a broad budding base (4 to 5 µm dia.)

  •          The budding cell usually remains attached to the parent cell, separating only when reaching the same size as the parent.

  •          Yeast cells have rather thick, refractile walls.

  •          Older cultures may produce thick-walled chlamydoconidia (7 to 18 µm dia.)

Confirmation of identification is made by demonstrating conversion between the dimorphic forms.  Commercial kits are available for identification using DNA probes or by testing for a specific exoantigen by immunodiffusion.  The appearance of Blastomyces dermatitidis may be confused with Scedosporium species or Chrysosporium species.

Blastomyces dermatitidis is very pathogenic and should be handled with extreme care in a Level 3 laboratory.

New Content (August 20th, 2014)
Added here are a few more photos taken from a new case.  The photos below are from a fine needle aspirate (FNA) specimen taken from the lungs.  Material shown is stained using the Periodic Acid Schiff (PAS) stain as well as the Gomori Methenamine Silver Stain (GMS).

 Blastomyces dermatitidis - numerous cells are seen within the FNA of material from the lung.  The insert shows one cell in particular and the large based budding as a daughter cell is being produced.
(1000X, PAS, Nikon)

Blastomyces dermatitidis - large, thick-walled cell shown in center of photo.  Pair of dividing cells seen at bottom of photo.
(1000X, PAS, Nikon)

Blastomyces dermatitidis -Budding cell show in center of photo.  Other single cells also present.
(1000X, GMS, Nikon)

Blastomyces dermatitidis - Several cells visible in this photo: most prominent is the large thick-walled cell in the lower center.
(1000X, Stain not noted (oops!), DMD-108)

Blastomyces dermatitidis - Large budding cell with insert showing the dimension of the base of the bud at this stage.
(1000+10X, Stain not noted (oops!), DMD-108)

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