CLS Blastomyces dermatitidis.pptx

fungus

1. BLASTOMYCOSIS ( Blastomyces dermatitidis ) Sundar Khadka , Microbiologist HIV Reference Unit, NPHL

2. Blastomycosis Blastomycosis is a chronic granulomatous and suppurative disease having a primary pulmonary stage that is frequently followed by dissemination to other body sites, chiefly the skin and bone. Although the disease was long thought to be restricted to the North American continent, in recent years cases have been diagnosed in Africa, Asia and Europe.

3. History Casper Gilchrist first reported the disease in 1894 from Baltimore in a patient with lesions involving the skin. Clinical entity designated in his honour as Gilchrist’s disease Chicago disease or North American Blastomyces Thermal basis of dimorphism first described by Hamburger in 1907 Dermatitidis - infection of skin

4. Habitat Acidic soils with rich organic debris such as decaying vegetative matter rotting wood feces of birds and bats(providing increased nitrogen content)

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6. Mycology General characteristics:   Dimorphic fungi that behaves in two different manners according to temperature of environment :   o        Mold-like at 25 C §         Branched hyphae are present   o        Yeast-like at 37 C §         Broad-based yeast buds are present

7. Mycology Dimorphic fungi Macroscopic morphology Filamentous fungus form: Grown at 25 to 30 o C: Exhibits a cottony or downy texture(covered with fine, soft hair or feathers) Colonies produce white Arial hyphae on the surface which may turn a yellowish to tan(pale brown) colour as the colony ages. The reverse is typically a light tan to brown. Moderately slow growth, usually maturing in about 2 weeks but suspect cultures should be held for 8 weeks

8. Microscopic Morphology Filamentous fungus form 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.

9. Yeast Form

10. Macroscopic Morphology   Yeast form Grown at 37 o C:  Slow to moderate growth Best chance for conversion :Blood Agar or Brain-Heart Infusion (BHI)) incubated in about 6% CO 2. yeast form cream to tan in colour , heaped or wrinkled, granular to verrucose ( like a wart or warts ). The yeast form of Blastomyces dermatitidis inhibited by cycloheximide .

11. Microscopic Morphology Yeast Form Grown at 37 o C: (enhanced by rich media and CO 2 , or in infected tissue) Yeast-like cells (~8 to 15 µm dia.) exhibit a broad budding base (4 to 5 µm dia.) broad‐based bud or “dumbbell-shaped” or letter 8 The budding cell usually remains attached to the parent cell, separating only when reaching the same size as the parent.

12. Yeast cells have thick , refractile walls. Older cultures may produce thick-walled chlamydoconidia (7 to 18 µm dia.) Note that this organism forms a broad‐based bud , whereas Cryptococcus neoformans is a yeast that forms a narrow‐based bud.

13. Serotypes Two types AK type: worldwide K type: prevalent in Africa and smaller in size

14. Pathogenesis of the Fungi Portal of entry primary mycoses – respiratory portal; inhaled spores subcutaneous - inoculated skin; trauma cutaneous and superficial – contamination of skin surface Humans are susceptible to acquiring infections by close contact with dogs or through scratches and bites.

15. Virulence factors – thermal dimorphism toxin production capsules and adhesion factors hydrolytic enzymes inflammatory stimulants

16. Pathogenicity Transmission: inhalation of conidia à yeast form once in lungs Once inside the lungs, the change of environment (carbon dioxide content, organic nutrients, pH and primarily the increased temperature (37 o C)), and causes the fungal spores to develop into the yeast cell form. The yeast form is disseminate to other areas of the body via blood Symptoms may not appear for up to 120 days post infection.

17. Inflammatory pyogranulomatous reactions occur at the initial pulmonary site and at the widespread foci of infection Pyogranulomatous is supparative of neutrophils followed by granulomas of mononuclear cells. Mixed neutrophilic and mononuclear cells response is distinctive of blastomycosis Necrosis and fibrosis may occur granuloma donot caseate

18. Sign and Symptoms Asymptomatic: 50% of infections Blastomycosis symptoms often mimic symptoms of upper respiratory infections The incubation period is 30 to 100 days, although infection can be asymptomatic   When individuals are exposed to  Blastomyces dermatitidis  the early symptoms may include: Approximately 25% of diagnosed Blastomycosis cases affect the lungs. While 35% of diagnosed cases involve both the lungs and skin   dry cough fever heavy sweating fatigue general feeling of ill health

19. Blastomycosis 1. Pulmonary Blastomycosis Acute Pulmonary: lobar or segmental consolidation, mimics bacterial pneumonia Chronic Pulmonary: lobar infiltrates mimics bronchogenic carcinoma 2. Cutaneous Blastomycosis Skin: ulcerative lesions, subcutaneous nodules that abscess 3.Osseous Blastomycosis osteomyelitis

20. 4. Disseminated Blastomycosis Bone / Joint: long bones, ribs, vertebrae osteolytic Genitourinary Tract: prostate and epididymis Other organs: CNS, pericardium, adrenal gland, GI 5. Miscellaneous types of Blastomycosis Extension of pulmonary Liver and spleen mostly affected Granulomatous lesions present

21. Skin lesions resulting from the dissemination of the fungus from the lungs

22. Diagnosis Laboratory specimens depend on the manifestation of the disease: pulmonary infection: sputum, BAL, Tracheal secretions, transbronchial biopsy skin lesions: skin scrapings or pus, biopsy from skin lesions Other specimens : biopsy of granulomatous lesions, Exudates, urine for antigen detection

23. Direct Demonstration KOH HE stain PAS stain GMS stain Broad based budding yeast cells

24. Fungus culture Media: Sabourauds dextrose agar, BHI agar, Blood agar , Blood – Glucose- cysteine agar at 25°C and 37° C On Sabourauds dextrose agar25°C to 30° C: Initially the colony appears yeast-like at room temperature and then develops hyphal projections, eventually becoming a fluffy white mould . In microscopic Examination, B. dermatitidis has round to pyriform , 4 to 5 μm conidia attached directly to the hyphae or on short stalks.

25. Confirmation of identification 1.demonstrating conversion between the dimorphic forms.(Mold to Yeast Conversion; M to Y) Media: Brain Heart Infusion agar, Blood agar, Blood-glucose- cysteine agar, Cottonseed agar, Cysteine heart agar with rabbit blood 2. identification using DNA probes 3. testing for a specific exoantigen by immunodiffusion .

26. demonstrating conversion between the dimorphic forms. The mold colony inoculated to the surface of blood agar and incubated for 3 -5 days at 37°C. prickly appearance colonies , suggesting an intermediate stage of conversion. lactophenol blue mount of a portion of one of the prickly colonies illustrates a short hyphal segment that is converting into spherical yeast forms. A few individual yeast forms are also present. These yeast forms are relatively large with broad-based attachments. This is characteristic of the yeast conversion forms of Blastomyces dermatitidis .

27. Skin test Serological test Immunodiffusion test (precipitin) Complement fixation (CF) test Enzyme Immunoassay (EIA). Immunodiagnosis

28. Antigen detection in urine Molecular Diagnosis DNA probes

29. Intraperitoneal injection of conidia and mycelial form produce infections in mice , guinea pigs, rats and hamsters Yeast form equally effective in causing extensive lesions and death of mice Animal Pathogenicity