Mycology Systemic Dimorphic Fungi

Name: Mycology Systemic Dimorphic Fungi
Uploaded: 2017-07-19T12:26:11+00:00
Duration: PTM33S11
Description: Mycology Systemic Dimorphic Fungi

Mycology Systemic Dimorphic Fungi
Please click audio icon to hear Carol’s narration Mycology Systemic Dimorphic Fungi Hi I’m Carol Larson, your guide thru this tutorial about the systemic dimorphic fungi. This lesson will be of value to you as you rotate thru the Mycology unit during 2nd semester. You will find an audio icon on each screen of this presentation. Please click on the icon to hear my narration for that specific screen. You may also follow along with the lecture handout entitled “Systemic Dimorphic Fungi” that you can print out from this blackboard lesson. Division of Medical Technology Carol Larson MSEd, MT(ASCP)

General Characteristics
Click icon for audio Systemic Dimorphic Fungi General Characteristics Growth rate: 2-4 weeks Identification Direct specimen examination Colony morphology Microscopic morphology Definitive diagnosis is prove dimorphism Mycelial stage at 25-30ºC Yeast / spherule stage in host at 35-37ºC The systemic dimorphic fungi are generally slow growers taking 2-4 weeks to grow. To identify these fungi, a combination of direct examination of specimens along with culture results are used. This group of fungi are dimorphic, meaning they have two morphological stages – a mycelial stage that grows at 25-30ºC on fungal culture media, and a parasitic stage (either a yeast or spherule) that is found in the human host. The yeast stage can be cultured in the lab if incubated at 35-37ºC on blood-enriched media. To definitively identify this group, we must demonstrate both stages and prove their dimorphism.

Epidemiology Primarily in North America or South America Found in:
Click icon for audio Systemic Dimorphic Fungi Epidemiology Primarily in North America or South America Found in: Soil Decaying vegetation Bird droppings Bat droppings Epidemiologically, the systemic dimorphic fungi are found primarily in North America with the exception of Paracoccidioides being found primarily in South America. These fungi are found in several sources including soil, decaying vegetation, and bird and bat droppings.

Clinical Significance
Click icon for audio Systemic Dimorphic Fungi Clinical Significance Pathogens Man incidental host Acquire by inhalation Develop respiratory infection Can disseminate and become systemic infection Clinical significance of the systemic dimorphic fungi is that they are pathogens and man is an incidental host. Man acquires the fungus by inhalation of conidia and then develops a respiratory infection. The fungi can then become systemic and involve the internal organs such as the lymph nodes, bone, subcutaneous tissue and skin. Often times the fungal infection may be asymptomatic. Immunocompromised and long-term corticosteroid therapy patients are most prone to infections by this group of fungi.

Clinical Significance
Click icon for audio Systemic Dimorphic Fungi Clinical Significance Histoplasmosis Histoplasma capsulatum Blastomycosis Blastomyces dermatitidis Coccidioidomycosis Coccidioides immitis Paracoccidioimycosis Paracoccidioides brasiliensis We are going to focus on four etiologic agents in the systemic dimorphic fungal group. Each fungus causes its own disease. The first etiologic agent is Histoplasma capsulatum and it causes Histoplasmosis. You will quickly see that each disease has the same name as the organism with a “mycosis” at the end. Second we will look at Blastomycosis caused by Blastomyces dermatitidis. Third is Coccidioidomycosis caused by Coccidioides immitis. These first three are included in your objectives. The fourth disease, Paracoccidioimycosis caused by Paracoccidioides brasiliensis, is not in your objectives but I wanted to provide you with complete notes with respect to the systemic dimorphic fungi.

For the systemic dimorphic fungi, what must be demonstrated in the lab to definitively identify them? The systemic dimorphic fungi have two forms (mold and yeast/spherule) and both must be seen in the laboratory either in culture or in direct examination of a specimen.

How are the systemic dimorphic fungal infections acquired?
By inhalation of the conidia and fungal elements. The infection begins in the lungs for all of the systemic dimorphic fungi.

Histoplasmosis Histoplasma capsulatum Pathogenesis
Click icon for audio Histoplasmosis Histoplasma capsulatum Pathogenesis 95% of cases are asymptomatic Infection begins in lung Invades reticuloendothelial system Can disseminate (especially in immunocompromised patients) We are first going to look at Histoplasmosis. The etiologic agent is Histoplasma capsulatum. Pathogenically, 95% of the cases are asymptomatic. Infection begins in the lung upon inhaling the conidia and eventually it invades the reticuloendothelial system. It can disseminate to the lymphatic tissues, liver, spleen, kidneys, meninges, and heart. Immunocompromised patients are most prone to these infections.

Laboratory Diagnosis Specimen Histoplasmosis Sputum Blood Bone marrow
Click icon for audio Histoplasmosis Laboratory Diagnosis Specimen Sputum Blood Bone marrow Urine Oral lesion scraping Lymph node biopsy Liver Specimens that can be collected are quite varied depending upon where the infection is located. Examples include sputum, blood, bone marrow, urine, oral lesion scraping, lymph node biopsy, liver, etc.

Laboratory Diagnosis Direct detection methods Histoplasmosis
Click icon for audio Histoplasmosis Laboratory Diagnosis Direct detection methods Giemsa or Wright’s stains Calcofluor white stain, histological stains Look for small intracellular yeast cells Specimens can be evaluated directly for the yeast form of Histoplasma capsulatum. Histoplasma is commonly found intracellular in mononuclear cells as pictured here. So stains that can be used for blood and bone marrow include the Giemsa and Wright’s stains. It is important that the yeast be distinguished from the parasites, Leishmania and Toxoplasma. Additional stains that can be used on tissue sections include the Calcofluor white stain, PAS, Methenamine silver, and H&E stains.

Mold - Histoplasma capsulatum
Click icon for audio Histoplasmosis Mold - Histoplasma capsulatum Media Sab’s and Inhibitory Mold agars Good growth at 2-4 weeks Mycosel agar – no growth Incubate 25-30ºC Ambient air Up to 4-6 weeks To culture the mold phase in the lab requires the use of general purpose fungal media such as Sabouraud’s and Inhibitory Mold agars. Good growth is obtained at 2-4 weeks. If many organisms are present in the specimen, you may see growth at soon as 3-5 days. Histoplasma will not grow on Mycosel agar. The culture media is incubated at 25-30ºC in ambient air for up to 4-6 weeks.

Click icon for audio Histoplasmosis Mold - Histoplasma capsulatum Colony morphology The colony morphology for Histoplasma capsulatum has a white colony that becomes brownish on the surface and yellowish on reverse. Its texture is wooly to granular.

Click icon for audio Histoplasmosis Mold - Histoplasma capsulatum Microscopic morphology The microscopic morphology for Histoplasma capsulatum is its diagnostic form. The hyphae are hyaline and septate. Its macroconidia are unicellular, hyaline, thick-walled, and tuberculate (meaning bumpy or knobby). Microconidia are unicellular, and hyaline, with a smooth or rough wall. Please keep in mind that Histoplasma can be confused with Sepedonium and to differentiate them remember that Sepedonium does not have a yeast phase.

Yeast - Histoplasma capsulatum
Click icon for audio Histoplasmosis Yeast - Histoplasma capsulatum Media Blood enriched media Incubate at 35ºC in ambient air for 2-4 weeks Colony morphology Microscopic morphology After the mold phase has been cultured in the lab, it is important to prove the fungus’ dimorphism. The mold is subcultured to a blood enriched media such as BHI and incubated at 35ºC in ambient air for 2-4 weeks. It is often very difficult to make the conversion from the mold phase to the yeast so some labs prefer to confirm the dimorphism by serological testing. Colony morphology for the yeast phase has a creamy texture with cream color on its surface and reverse. Microscopically the yeast morphology has small budding yeast that measure 2-4 um in length. Another clue is to correlate findings with the pathological reports of tissue biopsies that would have the intracellular yeast cells.

Serological Testing Exoantigen test Histoplasmosis Immunodiffusion
Click icon for audio Histoplasmosis Serological Testing Exoantigen test Immunodiffusion There is serological testing available to identify Histoplasma in the form of an exoantigen test performed by immunodiffusion.

What is the diagnostic form for Histoplasma capsulatum and what does it look like?
The mold phase – its macroconidia are large, round, unicellular, thick-walled and tuberculated.

In patient specimens, where is a common place that you would find Histoplasma capsulatum yeast?
Histoplasma capsulatum yeast cells are often found intracellular in mono-nucleated cells.

Blastomycosis Blastomyces dermatitidis Pathogenesis
Click icon for audio Blastomycosis Blastomyces dermatitidis Pathogenesis Acute or chronic suppurative and granulomatous infection Infection begins in lung Can disseminate to lungs, bone, soft tissue and skin Blastomycosis is an acute or chronic suppurative and granulomatous infection. Its etiologic agent is Blastomyces dermatitidis. Infection starts in the lungs by inhaling conidia or hyphal elements and it can spread to involve the lungs, long bones, soft tissue and skin.

Laboratory Diagnosis Specimen Blastomycosis Cutaneous lesions Sputum
Click icon for audio Blastomycosis Laboratory Diagnosis Specimen Cutaneous lesions Sputum Biopsy of affected organ Specimens of choice for isolating Blastomyces include cutaneous lesions, sputum, and a biopsy of the affected organ.

Laboratory Diagnosis Direct detection methods Blastomycosis KOH prep
Click icon for audio Blastomycosis Laboratory Diagnosis Direct detection methods KOH prep Histological stains Look for large spherical budding yeast with single bud connected to parent cell by a broad base The specimens can be examined directly using a KOH preparation or various histological stains such as PAS, Methenamine silver stain, and the H&E stain. These specimens are examined for spherical budding yeast that have a single bud that is connected to the parent cell by a broad base. The yeast is generally 8-15 um in size. Pictured here you can see the dark-red staining yeast cells in a tissue biopsy.

Mold - Blastomyces dermatitidis
Click icon for audio Blastomycosis Mold - Blastomyces dermatitidis Media Sab’s and Inhibitory Mold agars Good growth at 1-4 weeks Mycosel agar – no growth Incubate 25-30ºC Ambient air Up to 4-6 weeks The mold phase of Blastomyces dermatitidis can be cultured on Sabouraud’s and Inhibitory Mold agars. Good growth occurs at 1-4 weeks. Blastomyces will not grow on Mycosel agar. The culture media is incubated at 25-30ºC in ambient air for up to 4-6 weeks.

Mold - Blastomyces dermatitidis
Click icon for audio Blastomycosis Mold - Blastomyces dermatitidis Colony morphology Microscopic morphology Colony morphology of Blastomyces dermatitidis has a white to beige colony on its surface and the reverse is pale to brownish. The colony texture is downy. Microscopic morphology will have hyaline, septate hyphae. The conidiophores are short and unbranched. The conidia are hyaline, pyriform, unicellular, terminal, and solitary. Basically the mold form of Blastomyces is very nondescript.

Yeast - Blastomyces dermatitidis
Click icon for audio Blastomycosis Yeast - Blastomyces dermatitidis Media Cottonseed conversion agar Incubate at 35ºC in ambient air for 1 week Colony morphology Upon culturing the mold form in the lab, the yeast form must be demonstrated to determine the identification of Blastomyces dermatitidis. Cottonseed conversion agar can be used and it should be incubate at 35ºC in ambient air for 1 week. Blastomyces is the only instance where mold-to-yeast conversion is feasible in the clinical lab. Colony morphology for the yeast form has a white to beige colony that is creamy and granular to verrucose in texture.

Yeast - Blastomyces dermatitidis
Click icon for audio Blastomycosis Yeast - Blastomyces dermatitidis Microscopic morphology Broad-Based Budding yeast The microscopic morphology for Blastomyces dermatitidis is the diagnostic form. The yeast cells have thick refractile walls, and a single bud with a broad base. An easy way to remember this is a “Broad Based Budding yeast” has a lot of B’s in it and Blastomyces dermatitidis begins with B.

Other Test Methods Exoantigen test Nucleic acid amplification assay
Click icon for audio Blastomycosis Other Test Methods Exoantigen test Immunodiffusion Nucleic acid amplification assay Other test methods include the exoantigen test performed by immunodiffusion, and the nucleic acid amplification assay which is now replacing the serological test methods.

What is the diagnostic form for Blastomyces dermatitidis and what does it look like?
The yeast phase – it has a broad based single bud and is large in size.

Describe the appearance of the mold form of Blastomyces dermatitidis?
The mold form of Blastomyces is very nondescript. It has hyaline, septate hyphae. The conidiophores are short and unbranched and the conidia are hyaline, pyriform, unicellular, terminal, and solitary.

Coccidioidomycosis Coccidioides immitis Pathogenesis
Click icon for audio Coccidioidomycosis Coccidioides immitis Pathogenesis 60% of infections are asymptomatic Infection begins in lung Can disseminate (1%) Southwest U.S. desert area (endemic to San Joaquin Valley) Coccidioidomycosis is an infection most often seen in the southwest United States desert areas. It is endemic to the San Joaquin Valley and is often referred to as San Joaquin Valley Fever. Its etiologic agent is Coccidioides immitis. Pathogenically, 60% of the infections are asymptomatic and have a self-limited respiratory tract infection. Infection begins in the lungs by inhaling the mold’s arthroconidia. Coccidioides is very infectious and one only needs to inhale 10 conidia to become infected. The disease can disseminate (in about 1% of the cases) with extension to visceral organs, meninges, bone, skin, lymph nodes, and subcutaneous tissue.

Laboratory Diagnosis Specimen Coccidioidomycosis Sputum
Click icon for audio Coccidioidomycosis Laboratory Diagnosis Specimen Sputum Biopsy of affected organ Specimens of choice include sputum and a biopsy of the affected organ.

Laboratory Diagnosis Direct detection methods Coccidioidomycosis
Click icon for audio Coccidioidomycosis Laboratory Diagnosis Direct detection methods KOH prep, Calcofluor white stain Histological stains Look for spherule No yeast form Upon performing a direct examination of the patient’s specimen, a thick-walled spherule measuring um in diameter, and containing either granular material or numerous small nonbudding endospores will be seen. Coccidioides does not have a yeast form. Various direct exam methods that can be used include a KOH preparation, a Calcofluor white stain, and various histological stains such as PAS, Methenamine silver, and H&E stains.

Mold - Coccidioides immitis
Click icon for audio Coccidioidomycosis Mold - Coccidioides immitis BIOHAZARD to lab personnel Media BAP, Sab’s, Inhibitory Mold, and Mycosel Good growth at 3 days to 3 weeks Incubate 25-30ºC Ambient air Up to 4 weeks Coccidioides immitis is a major BIOHAZARD to lab workers, so it is very important to use strict safety precautions when examining fungal cultures when suspecting Coccidioides. You must do all of your work in a biological safety hood, wear a gown, gloves and mask, and seal all culture plates with tape. Media used to culture Coccidioides includes Sabouraud’s, Mycosel and Inhibitory Mold agars. Good growth occurs as soon as 3 days or can take up to 3 weeks. There is no growth on a blood agar plate. The media is incubated at 25-30ºC in ambient air for up to 4 weeks.

Click icon for audio Coccidioidomycosis Mold - Coccidioides immitis Colony morphology Colony morphology for Coccidioides immitis has white colonies (or sometimes the color can be beige, pink, cinnamon, yellow or brown). The reverse is pale, or sometimes can be orange, or pale to dark brown. Its texture is wooly to glabrous. Please notice how this culture plate has tape to keep it sealed. Safety is extremely important!

Click icon for audio Coccidioidomycosis Mold - Coccidioides immitis Microscopic morphology Coccidioides immitis microscopically has hyaline, septate hyphae. Conidiophores are absent and you will predominantly see arthroconidia. The arthroconidia are unicellular, rectangular to barrel shaped, often somewhat wider in diameter than the hyphae, and they are alternating with empty cells called disjunctors. Freed arthroconidia possess annular frills which are persistent remnants of the wall of the broken disjunctor. This mold phase will grow at both 25ºC and 35ºC.

Spherule - Coccidioides immitis
Click icon for audio Coccidioidomycosis Spherule - Coccidioides immitis Not recommended Media Special media incubated at 42ºC Can infect lab animals and do tissue biopsy (experimental) Remember that there is no yeast phase, so to prove dimorphism, the presence of spherules seen in a direct specimen examination aids in this proof. To culture the spherule, special media must be used and incubated at 42ºC. This is not recommended as proof for dimorphism. Another method is to experimentally infect lab animals and perform a tissue biopsy, but again this is costly and not recommended.

Other Test Methods Exoantigen test Nucleic acid amplification assay
Click icon for audio Coccidioidomycosis Other Test Methods Exoantigen test Immunodiffusion Nucleic acid amplification assay The exoantigen test and nucleic acid amplification assay are good alternatives to determining Coccidioidomycosis.

What form for Coccidioides immitis is found in patient specimens?
The spherule. There is no yeast phase for this fungus.

What does Coccidioides immitis look like when cultured on SAB agar that has been incubated at 30°C?
Barrel-shaped alternating arthroconidia.

Paracoccidioidomycosis
Click icon for audio Paracoccidioidomycosis Paracoccidioides brasiliensis Pathogenesis Infection begins in lung Can be asymptomatic and then disseminate to nasal and oral mucosa, gingivae, or conjunctivae South America (South American Blastomycosis) You objectives do not include Paracoccidioidomycosis, but I want to briefly touch on it. Its etiologic agent is Paracoccidioides brasiliensis. As with the other systemic dimorphic fungi, infection begins in lungs by inhaling conidia from the environment. The patient can have mucosal lesions possibly caused by trauma to the oropharynx caused by chewing infested vegetation. The disease can be asymptomatic and then disseminate to the nasal and oral mucosa, gingivae, or conjunctivae. It can also disseminate to the lymphatic system, spleen, intestines, liver, brain, meninges, and adrenal glands in some patients. This disease is seen most often in South America (and mainly in Brazil, Venezuela, and Colombia).

Laboratory Diagnosis Specimen Paracoccidioimycosis Sputum
Click icon for audio Paracoccidioimycosis Laboratory Diagnosis Specimen Sputum Biopsy of affected organ Specimens of choice include sputum and a biopsy of the affected organ.

Laboratory Diagnosis Direct detection methods Paracoccidioimycosis
Click icon for audio Paracoccidioimycosis Laboratory Diagnosis Direct detection methods KOH prep, Calcofluor white stain, and Histological stains Look for large round or oval, multiple budding yeast cells (“mariner’s wheel”) What you will see on a direct specimen exam is a large, round or oval, multiple budding yeast measuring between 8 and 40 um in diameter. It resembles a “mariner’s wheel”. This is the diagnostic form for Paracoccidioides brasiliensis. An easy way to remember this is that the organism name has “brazil” in it and to get to Brazil you need to take a boat – thus the mariner’s wheel. Methods used to detect this structure in specimens includes a KOH preparation, Calcofluor white stain and histological stains such as PAS, Methenamine silver, and H&E stains.

Mold - Paracoccidioides brasiliensis
Click icon for audio Paracoccidioimycosis Mold - Paracoccidioides brasiliensis Media Sab’s and Inhibitory Mold agars Good growth at 3-4 weeks Mycosel agar – no growth Incubate 25-30ºC Ambient air Up to 4-6 weeks Culturing Paracoccidioides brasiliensis in the lab is very similar to the other dimorphic fungi as you can see here.

Mold - Paracoccidioides brasiliensis
Click icon for audio Paracoccidioimycosis Mold - Paracoccidioides brasiliensis Colony morphology Microscopic morphology Colonies for Paracoccidioides are slow growing and their morphology has white, pink, beige or brown colonies with the reverse being yellowish to brown. The texture is glabrous to velvety. Microscopically its morphology is very nondescript with hyaline, septate hyphae (that is often sterile); conidiophores are absent; and aleurioconidia, arthroconidia and chlamydospores can be present.

Yeast - Paracoccidioides brasiliensis
Click icon for audio Paracoccidioimycosis Yeast - Paracoccidioides brasiliensis Media Blood enriched media Incubate at 35ºC in ambient air for 4 weeks Colony morphology Microscopic morphology The yeast form for Paracoccidioides will grow on blood enriched media such as BHI that is incubated at 35ºC in ambient air for 4 weeks. It grows very slowly and culture is not the best way to prove dimorphism. Colony morphology shows a white creamy colony. Microscopic morphology has the characteristic yeast cells with multiple “satellite” budding that looks like the mariner’s wheel. This is the diagnostic form (but is usually seen in direct specimen exams.)

Other Test Methods Exoantigen test Paracoccidioimycosis
Click icon for audio Paracoccidioimycosis Other Test Methods Exoantigen test Immunodiffusion The exoantigen test is available for identification.

What is the diagnostic form for Paracoccidioides brasiliensis and what does it look like?
The yeast form appears as a large multiple budding yeast that looks like a mariner’s wheel.

In Summary … Diseases Identification Systemic Dimorphic Fungi
Click icon for audio Systemic Dimorphic Fungi In Summary … Diseases All begin infection in lungs Disseminate Identification Demonstrate dimorphism Direct specimen exam Culture (mold and yeast forms) Other testing The systemic dimorphic fungi cause diseases that start in the lungs due to the inhalation of the fungus and then disseminate to various organs throughout the body. We looked at Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis, and Paracoccidioides brasiliensis. To identify these fungi, we must demonstrate in the lab both forms of the fungus to prove that it is dimorphic. This can be done through the use of direct specimen exams, culture of the mold and yeast forms, and serological and molecular testing.

Who am I? Growth at 25ºC Sputum – silver stain Coccidioides immitis

Blastomyces dermatitidis
Who am I? Growth at 25ºC Growth at 35ºC Blastomyces dermatitidis

Histoplasma capsulatum
Who am I? Growth at 25ºC Wright’s stain of blood Histoplasma capsulatum

Mycology Systemic Dimorphic Fungi

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