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| Topic 6 | [MLS 415] Mycology and Virology M1. Systemic Mycoses Professor: Joanne Krystianne Tago Date: March 3, 2024 Primary Systemic Mycoses ENDEMIC MYCOSES → particularly “Primary” Systemic Mycoses – to differentiate it from other pathogenic fungal elements because some species can also cause d...

| Topic 6 | [MLS 415] Mycology and Virology M1. Systemic Mycoses Professor: Joanne Krystianne Tago Date: March 3, 2024 Primary Systemic Mycoses ENDEMIC MYCOSES → particularly “Primary” Systemic Mycoses – to differentiate it from other pathogenic fungal elements because some species can also cause disseminated systemic infections Coccidioidomycosis Histoplasmosis Blastomycosis Paracoccidioidomycosis Caused by DIMORPHIC FUNGI YEAST PHASE On Enriched media w/ Blood, 35 – 37 deg C Seen in vivo, aka “tissue/invasive phase” Coccidioidomycosis MYCELIAL PHASE On Sabouraud Dextrose Agar at 25 – 30 deg C Saprophytic, observed in vitro Transmission Inhalation of Fungal Spores (Most common) Initial: Pulmonary Infection (Symptomatic/Asymptomatic) o symptomatic/asymptomatic o if untreated, it will develop into a disseminated forms of infection or progressive infections o have severe symptoms brought about by the spread of organisms from the lungs to distant organs Dissemination (in HIV or prolonged corticosteroid treatment) o mostly seen in immunocompromised individuals − HIV or under prolonged corticosteroid treatment Geographic Restrictions o Endemic because causative agents have a specific location where they thrive Serological Tests: o (1) Complement Fixation Test, (2) Immunodiffusion Test, (3) Tube precipitin Test, (4) Enzyme Immunoassay Most infections are asymptomatic or mild and resolve without treatment however, a small but significant number of patients develop pulmonary disease, which may involve dissemination from the lungs to other organs - (Arthroconidia of Coccidioides spp.) Also known as: o Valley fever o San Joaquin Valley Fever o Desert fever/ desert rheumatism MOT: Inhalation of arthroconidia o some cases were caused by puncture with infected objects or organ transplantation, and sexual contact (very rare) 60% = asymptomatic and self-limited Respiratory Tract Infections (Males are more susceptible) 40% = Flu-like condition DISSEMINATION Visceral organs Meninges Bone Skin Lymph nodes Subcutaneous tissue The disease occurs more frequently in certain racial groups (commonly on dark-skinned individual); in decreasing order: 1. Filipinos, 2. African Americans, 3. Native Americans, 4. Hispanics, and 5. Asians Biology of Coccidioidomycosis The initial host defenses are provided by the alveolar macrophages, which are usually capable of inactivating the conidia and inducing a robust immune response this process typically leads to granulomatous inflammation and the production of both antibodies and cell-mediated immunity Source: Centers for Disease Control and Prevention Found in Soil or Dusty environment @mlstranses | 1 In the environment (Desserts), Coccidioides ssp. exists as a: 1. Mold with septate hyphae 2. The hyphae fragment into arthroconidia which measure only 2-4 um in diameter. 3. Arthroconidia are EASILY AEROSOLIZED when disturbed. 4. Arthroconidia are inhaled by a susceptible host (4) and settle into the lungs. 5. The new environment (37 deg C) signals a morphologic change, and the arthroconidia become SPHERULES. 6. Spherules divide internally until they are filled with endospores (6). 7. When a spherule ruptures (7) the endospores are released and disseminate within surrounding tissue. 8. ENDOSPORES are then able to develop into NEW SPHERULES and repeat the cycle. Coccidioides spp. BOTH ARE MORPHOLOGICALLY INDISTINGUISHABLE o Coccidioides immitis = “California” spp. o Coccidioides posadasii = “Non-California” spp. ▪ Common in Mexico ▪ Slow growing on media with high salt concentration (Halophilic) DISSEMINATED COCCIDIOIDES Life Cycle of Coccidioides (Source: Barker, n.d., Obtained from ResearchGate) When a conidia lands a favorable environment, it will form into hyphal fragments 2. these hyphal fragments will branch out and entangle producing mycelia 3. the hyphal fragments in the mycelia will produce arthroconidia, which will break to produce the single arthroconidia 4. When the soil that contains the arthroconidia is disturbed, the arthroconidia may become dispersed in the air 5. when accidentally inhaled by an individual, the arthroconidia will morphologically change into its tissue or invasive phase due to temperature change ⇒ spherule 6. sporangiospores or endospores are then formed within the spherule by repeated cytoplasmic cleavage 7. the spherule will become engorged 8. after 120 hours, the spherules rupture releasing 9. endospores 10. the endospores may either be: a. released outside the body and proceed with saprobic life cycle b. remains within the body and reinitiate the cycle of spherules development 1. [L]: Chronic cutaneous coccidioidomycosis ; [R]: Extension of Pulmonary Coccidioidomycosis (Armpits) Begins with pulmonary infection → Severe cutaneous involvement (Granulomatous lesions) It may also show a large superficial ulcerated plaques under the armpits and upper chest; involvement of muscles and subcutaneous tissue and skin after pulmonary infection ○ There is also a presence of draining sinuses (Holes seen in the R image) Coccidioides immitis – LABORATORY DIAGNOSIS 1. Blood testing, PCR, Chest X-ray 2. Direct Examination a. Finding of spherules in sputum, draining sinuses or tissue specimen b. 10-20% KOH; Calcofluor White Microscopic Characteristics of Coccidioides immitis MYCELIAL PHASE YEAST/TISSUE FORM Septate, branched hyphae Non-budding, thick walled Thick-walled barrel-shaped, spherule rectangular arthroconidia that Outer wall: Mannan, protein, alternate with empty cells called lipid disjuncture cells Inner wall: Chitin, 3-O-Methyl mannose 20-200 um in size @mlstranses | 2  With numerous non-budding spores small, Confirmatory Tests for Coccidioidomycosis IMMUNODIFFUSION TEST POSITIVE RXN: Precipitin line/band formation Detects coccidioidal IgM (precipitins) & IgG (CF) Indicates ACTIVE primary infection Macroscopic Characteristics of Coccidioides immitis Control 1 IgM-positive serum Control 2 IgG-positive serum Antigen 1 antigen reactive to IgM antibodies Antigen 2 antigen reactive to both IgM and IgG antibodies Observe rxn after 24 hours; if the control has no lines observed repeat the test Serological Test for Coccidioidomycosis: COMPLEMENT FIXATION TEST 3-21 days Sabouraud Dextrose Agar (SDA), Inhibitory Mold Agar (IMA) Delicate, fluffy white, which turns tan or brown with age. In vitro production of spherules: Incubation in a complex medium at 40 degrees Celsius with 20% Carbon dioxide (CO2) or by animal inoculation; to shift from mold to yeast CAUTION for Coccidioides immitis The MOST INFECTIOUS of all fungi Formerly a “Select agent” – biological agent that has the potential to pose a severe threat to public health and safety HANDLING: Use a Biosafety Cabinet for all preparation and handling Cultures MUST be sealed in a tape Cotton-plugged tubes is DISCOURAGED o Screw-capped tubes is needed Autoclave all cultures after identification is done! 1. 2. Skin Tests for Coccidioidomycosis Coccidioidin a. Crude toluene extract of mycelial culture i. rgnt contains arthroconidia b. (+) Induration of less than 5 mm Spherulin a. Prepared from cultured spherules b. More sensitive, but less specific i. not specific because there are other organism that produces spherules (e.g. R. seeberi) Sample: Patient’s serum Reagents: commercially known coccidioidin antigen commercially known complement sheep’s RBCs coated with antibodies (indicator) This test relies on two intrinsic properties of complement complement will lyse sensitized RBCs complement will bind to the Fc portion of the IgG antibodies bound to antigens Result absence of hemolysis presence of hemolysis Interpretation POSITIVE (+) NEGATIVE (-) Treatment for Coccidioidomycosis SUPPORTIVE CARE o Self-limiting, mild symptoms MODERATE CASES o Fluconazole or Itraconazole SEVERE CASES o Posaconazole or Amphotericin B o Treatment for the lesions @mlstranses | 3  1. Histoplasmosis Most prevalent pulmonary mycosis MOT: Inhalation of conidia or small hyphal fragments Chronic granulomatous infection Begins in the lungs with Tuberculosis-like infections o Resembles S/s of TB Mostly asymptomatic and self-limited May disseminate and Re-infection may occur Sources of Infection: Histoplamosis Silos, air-conditioning units contaminated with BIRD DROPPINGS o birds are not infected, but their excrement provides superb culture conditions for growth of the fungus Accumulations of guano (debris of bats) in caves, attics, or parks Nitrogen-rich soils (where they usually thrive) o bat and bird poop increases the nitrogen levels in soil Clinical Manifestations of Histoplasmosis ACUTE PULOMARY HISTOPLASMOSIS b. usually seen on immunocompromised individuals OCULAR HISTOPLASMOSIS a. b. c. Loss of central vision – retina is the most affected Initial symptoms may be loss of central vision, blind spots, or scars on the retina Late stages – abnormal blood vessels in the eye may develop and cause changes in the vision Histoplasma capsulatum as the causative agent of Histoplasmosis a. b. 2. Self-limiting Fever, malaise, cough, headache, pain chills, and myalgias c. miliary: presence of grain-like nodules CHRONIC PULMONARY HISTOPLASMOSIS 4. 1. 2. 3. 4. 5. 6. a. b. 3. Opportunistic complication of Chronic Obstructive Pulmonary Disease (COPD) with emphysema and abnormal pulmonary spaces Silent killer – it would start really early and only discovered after 5-10 years DISSEMINATED HISTOPLASMOSIS In the environment (Caves, attics, silos), Histoplasma capsulatum exists as a mold with aerial hyphae. The hyphae produce macroconidia and microconidia spores that are aerosolized and dispersed. Microconidia are inhaled into the lungs by a susceptible host. The warmer temperature inside the host signals a transformation to an oval, budding yeast. The yeast are phagocytized by immune cells and transported to regional lymph nodes. From there they travel in the blood to other parts of the body (through the circulatory system) Laboratory Diagnosis of Histoplasmosis Direct Microscopic Examination Difficult to see in the sputum and other tissues Rarely in peripheral blood Bone marrow smear: Wright or Giemsa (BEST) Intracellular yeast in macropahges Dermatomyositis-like pattern; tubules appear like tuberculoid form of leprosy a. Yeast cells may be disseminated while inside macrophages @mlstranses | 4 Macroscopic Characteristics of Histoplasma capsulatum Microscopic Characteristics of Histoplasma capsulatum MYCELIAL PHASE YEAST PHASE Septate hyphae 2-5 u (microns) Large spherical or From a mold tuberculate Brain-Heart Infusion macroconidia Agar at 37 degrees Small, round, smooth Celsius microconidia Small, ellipsoidal Slow-growing (2-4 weeks) Sabouraud Dextrose Agar/ Inhibitory Mold Agar (MOLD): ○ White → brown, fine fluffy; white, yellow, or tan on reverse side when cultured, demonstrates 2 colonial forms ○ colony A: albino-type (white) ○ colony B: slightly tinged/colored (brown or cream or tan) both colony types can form identical yeast and tissue forms − but colony B is more pathogenic for mice and rabbits and can produce more macroconidia Brain Heart Infusion Agar (YEAST): Moist, white to cream heaped colony Histoplasmin Skin Test Produced by growing the mycelium in asparagine broth medium Positive Reaction: indicated by an area of induration >5mm in diameter after 48 hours Serology Tests for Histoplasma Capsulatum Detection of specific antibodies ○ Complement Fixation Test ○ Immunodiffusion Test ○ Direct Fluorescent Antibody Test ○ Counterimmunoelectrophoresis Test Closer look at Yeast phase of Histoplasma capsulatum. Laboratory Diagnosis of Histoplasma capsulatum Culture: LYSIS CENTRIFUGATION METHOD Used to enhance the recovery of H. capsulatum 10 ml blood w/ anticoagulants & reagents to lyse RBCs; centrifuge, Pellets with yeasts are inoculated in culture medium Treatment for Histoplasma Capsulatum Acute Pulmonary Histoplasmosis Management: ○ Supportive therapy and rest are the primary approaches. Treatment for Mild to Moderate Infection: ○ Itraconazole is the recommended treatment. Disseminated Disease Treatment: ○ Amphotericin B is the systemic treatment of choice for disseminated histoplasmosis. ○ This treatment is often curative, but patients may require prolonged treatment and monitoring for relapses. @mlstranses | 5  AIDS Patients Treatment Approach: ○ AIDS patients may experience relapses despite curative therapy in other individuals. ○ Maintenance therapy with itraconazole is necessary for AIDS patients to manage and prevent relapses. a. b. Traumatic autoinoculation Contamination of an open wound with infectious material 2. CHRONIC CUTANEOUS BLASTOMYCOSIS Blastomycosis Most common on exposed skin surfaces (face, hands, wrists, lower legs) 3. DISSEMINATED BLASTOMYCOSIS Chronic suppurative and granulomatous infection ○ With pus and granules ○ Common with Dogs and Cats (Zoonotic) inhalation of the conidia, hyphal fragments Lungs; long bones, soft tissue and skin ○ Dissemination may occur to any organ but preferentially to the skin and bones Serology Tests: Complement Fixation, Immunodiffusion, Enzyme Immunoassay Test Causative agent: Blastomyces dermatitidis Biology of Blastomycosis Extrapulmonary sites most common disseminated form: cutaneous type Skin, bones, Genitourinary tract, Central nervous system, and spleen 4. PULMONARY BLASTOMYCOSIS Most common from Pulmonary lesion heals by fibrosis and resorption ○ Scarring is prominent compared to TB Rarely calcifies 1. 2. 3. 4. 5. 1. In the environment (Rainforests), Blastomyces exists as mold with septate aerial hyphae. The hyphae produce spores. These spores are either inhaled, or inoculated into the skin of a susceptible host. The warmer temperature inside the host signals a transformation into a broad-based budding yeast. The yeast may continue to colonize the lungs or disseminate in the bloodstream to other parts of the body, such as the skin, bones and joints, organs, and central nervous system. Clinical Manifestations of Blastomycosis PRIMARY CUTANEOUS BLASTOMYCOSIS Laboratory Diagnosis of Blastomyces dermatitidis MICROSCOPY calcofluor white or KOH prep of pus, exudate, sputum yeast cells are large and have thick walls MYCELIAL PHASE Delicate, septate hyphae Round or pyriform conidia borne signify on conidiophores resembling “lollipops” Has shorter conidiophore than S. boydii ; Described as PEDUNCLE YEAST PHASE Biopsy tissues or body fluids: Hematoxylin & Eosin stain Large-spherical, 8-15 microns (u) Thick-walled yeast A single bud connected to its parent cell by a broad base @mlstranses | 6 ○ Adult type (Pulmonary & Disseminated) affects the lungs, lesions in the mouth and tongue Microscopic Characteristics of Paracoccidioides brasiliensis MYCELIAL PHASE Macroscopic characteristics of Blastomyces dermatitidis Small, sepate, branched hyphae with intercalary (in between septum) & terminal chlamydospores YEAST PHASE Large, round, or oval cells Multiple buds attached to the parent cell by a narrow base “Mariner’s wheel” Chlamydospores can grow at the tip of the hyphae or in between septum 7-21 days Sabouraud Dextrose Agar/Inhibitory Mold Agar ○ White, waxy, yeast-like, becoming cottony with white aerial mycelium; turns tan to brown with age Brain-Heart Infusion Agar with Blood ○ Cream to tan, waxy, wrinkled Serology Tests for Blastomyces dermatitidis Measurement of Complement Fixation Antibodies has not yet been proven reliable Yeast phase provides a more specific Antigen Immunodiffusion test = more sensitive and more specific than Complement Fixation Test Difference between H. capsulatum and P. brasiliensis lies within their forms – mariners wheel appearance of P. brasiliensis is in the yeast phase while for the H. capsulatum its in the mycelial phase Macroscopic Characteristics of Paracoccidioides brasiliensis Treatment for Blastomyces dermatitidis Itraconazole (Oral) and Amphotericin B (Intravenous) Paracoccidioidomycosis Chronic granulomatous infection Begins as a primary pulmonary infection MOT: Inhalation of fungal structures Asymptomatic but may disseminate Infects: Nasal or oral cavity, gingiva, and conjunctiva Do not spread from person-to-person Complement fixation, Immunodiffusion test Clinical Manifestations of Paracoccidioidomycosis Asymptomatic Form (Most of the time) ○ Initial form ○ Latent Symptomatic Form ○ Juvenile type swollen lymph node and skin lesions Very slow-growing Sabouraud Dextrose Agar ○ White, glabrous, leathery colony; tan-brown with age Brain Heart Infusion (BHI) ○ Cream to tan, moist, wrinkled colony which turns waxy with age Serologic Tests for Paracoccidioides brasiliensis infxn Immunodiffusion Test is extremely useful Complement Fixation Test is quantitative and useful for assessing prognosis, but cross-reactions occur with other fungi @mlstranses | 7 [MLS 415] Mycology and Virology M2: Opportunistic Mycoses Professor: Joanne Krystine Tago Date: March 8, 2024 TYPES OF OPPORTUNISTIC MYCOSES Candidiasis, systemic Cryptococcosis Aspergillosis Zygomycosis/ Mucormycosis Hyalohyphomycosis Pneumocystosis OPPORTUNISTIC MYCOSES Fungal infections in the body which occur almost exclusively in immunocompromised individuals The causative agents are common and found around us → usually have very low inherent virulence ○ Not asymptomatic; Not self-limiting ○ Usually infected are IMMUNOCOMPROMISED Candidiasis Most frequently encountered Endogenous in origin or nosocomial Fungemia & disseminated infection Candida spp. = normal microbiota in the oropharynx, gastrointestinal tract, genitourinary tract, skin In healthy individuals, Candidiasis are usually due to impaired epithelial barrier Systemic type of Candidiasis are usually seen on patients with cell-mediated immune deficiencies Predisposing Factors - Candidiasis Alteration in the normal skin and mucous membrane barriers (most common for immunocompetent individuals) Prolonged antibiotic administration Use of immunosuppressive drugs Diseases of the immune system Etiologic Agents - Candida spp. Candida albicans (most common) Candida parapsilosis Candida tropicalis Candida glabrata Antigenic Structures - Candida spp. 2 serotypes: A and B Proteases – breakdown of protein structures Enolase – glycolytic enzyme; immunodominant antigen in disseminated infections Heat shock proteins – family or proteins produced by cells usually in response to exposure to stressful conditions (extreme heat, cold or overexposure to UV light) ○ Discovered during wound healing and tissue repair Clinical Manifestations - Candida spp. OROPHARYNGEAL CANDIDIASIS (common) ○ seen on 5% newborns, 10% elderly patients ○ commonly seen on immunocompromised patients with Diabetes mellitus, leukemia, lymphoma, neutropenia, HIV ○ rarely seen healthy adults ○ includes: oral thrush; glossitis (inflammation of the tongue) or stomatitis (inflammation of mouth and lips) ○ the milky-like curd can be found in the buccal mucosa, tongue, gums, and sometimes far back into the pharynx may have a burning sensation, dryness of the mouth, loss of taste, problem in swallowing and drinking Perleche Oral thrush Vulvovaginitis “Diaper rash” in children Eye infections CUTANEOUS CANDIDIASIS ○ Intertrigo ⎼ commonly seen on folds of skin such as the axillary area, groin, submammary folds etc; skin barrier is damaged due to constant exposure to heat and friction @mlstranses | 1 ○ ○ Chronic Candida Onychomycosis – infection usually occur underneath the nails causing it to weaken and produce too much nail debris nail almost separates the nail bed nails are disfigured; crumpled up towards the center and obvious thickening usually seen on: DM, hypothyroidism, Addison's disease, patients with malfunctional thyroid, malnutrition, and malignancies Paronychia ⎼ infection of the fingernails, especially around the cuticle area commonly seen on individuals who constantly have their fingers wet or constant contact with flour (for individuals who work on bakeries) Intertrigo Chronic Candida Onychomycosis [UPPER L to R] PAS and Calcofluor white [LOWER L to R] IF and GMS MACROSCOPIC 24-48 hours (rapid growers) raised, cream-colored, opaque, 1-2 mm medium hyphae may be observed after several days CHROMAGAR CANDIDA (apart from SDA) ○ useful in differentiation Candida spp. as they are allowed to produce different colors ○ C. albicans ⎼ green; C. tropicalis ⎼ blue C. glabrata ⎼ purple; C. krusei ⎼ pink Paronychia Laboratory Diagnosis SPECIMEN skin and nail scrapings urine, sputum, bronchial washing, CSF or pleural fluid, blood Tissue samples (for cutaneous types) DIRECT MICROSCOPIC EXAM 10% KOH, Parker ink or calcofluor white budding yeast cells (blastoconidia) pseudohyphae: strongly Gram (+) CORNMEAL AGAR WITH TWEEN80 (CMA-T80) Identification of Candida spp.a nd other yeasts (pseudohyphae, chlamydospores, blasto-, & arthroconidia) ○ Tween 80: stimulates the conidiation by reducing the surface tension of the culture media DALMAU Expected results: C. albicans ○ with: chlamydospore & blastoconidia arranged along pseudohyphae DALMAU PLATE TECHNIQUE A procedure on planting organisms on either rice meal agar or corn meal agar to allow conidiation Procedure: 1. divide the rice agar plate into sections 2. label the sectors with the number of the tested strain 3. inoculum from the primary media will be inoculated into the rice meal agar 4. after initial streaking, make 4 cuts @mlstranses | 2 C. neoformans - Round to oval Rare, usually not separated by capsule seen Saccharomyces - Large and spherical Rudimentary H sometimes present Numerous, Maybe present but Septated hyphae is resemble difficult to find present Geotrichum Trichosporon Commonly encountered yeast in CMA-T80 Agar 5. 6. 7. Cover with a coverslip the cut portion to introduce a microaerophilic environment Incubate the agar plates aerobically @ 25-30 deg C for 24-48 hours Examine the agar plate under the microscope GERM TUBE hypha-like extension with no constriction at the point of origin ○ look likes it was pulled away from the mother cell formed upon incubation w/ serum at 37°C for 1-3 hours similar to pseudohyphae but it has construction CRYPTOCOCCOSIS An acute, subacute or chronic fungal infection that has several manifestations infections are mild or asymptomatic, or does not require antifungal treatment for immunocompetent individuals Disseminated Disease: → Meningitis In 2/3 of patients. Cryptococcal meningitis could have microscopic lesions on the brain; life-threatening and requires aggressive therapy commonly in patients with AIDS Acquired through inhalation Found in soil Etiologic Agents - Cryptococcus spp. C. neoformans primarily present in soil contaminated with bird dropping, particularly of pigeons C. gattii usually present in soil around trees saprophyte bat, or bird (pigeon) droppings, decaying vegetations, fruit, plants MOT: inhalation (lungs) disseminated (meninges & other sites) endocarditis, hepatitis, renal infection, pleural effusion Attacks IMMUNOCOMPROMISED individuals Organism Arthroconidia Blastoconidia Pseudohyphae or Hyphae C. albicans - Spherical clusters at regular intervals on pseudohyphae Chlamydoconidia on hyphae C. glabrata - Small, spherical, tightly compact None C. krusei - Elongated. clustered at Branched septae of pseudohyphae pseudohyphae C. parapsilosis - Present but not characteristic Sagebrush like. Giant hyphae Organism Arthroconidia C. kefyr (pseudotropicalis) - Elongated, parallel to PH present, not pseudohyphae characteristic C. tropicalis - Randomly appear on PH present, not PH & H characteristic Blastoconidia Laboratory Diagnosis (C. neoformans) DIRECT MICROSCOPY → specimen depends on the infected part spherical, single or multiple budding, thick walled yeast cell (2 to 15 um) wide, refractile polysaccharide capsule CAPSULE ○ India ink preparation of CSF (visualization of the thick cell wall) ○ Mucicarmine ○ Masson-Fontana Pseudohyphae or Hyphae [LEFT] Pleural fluid on KOH [RIGHT] Capsule on India ink @mlstranses | 3 MACROSCOPIC Colonies in 1-5 days (rapid growers) smooth, white to tan, mucoid, gelatin-like colonies (soap-bubble) brown-black colonies on Niger Seed Agar ○ their ability to produce melanin is enhanced vue to the enzyme, phenoloxidase SDA without cycloheximide ○ Cryptococcus is sensitive to cycloheximide membrane filter technique (optimal recovery) ○ for watery/fluid-like specimen Organism FERMENTATION Urease Nitrate Reducti on G M S L C. albicans + + - - - - C. tropicalis + + + - - - C. parasilosis + - - - - - C. glabrata + - - - - - C. neoformans - - - - + - Geotrichum - - - - - - T. beigelii - - - - + - [LEFT] SDA without cycloheximide [RIGHT] Niger Seed Agar Identification of Yeast Cells Latex test for cryptococcal capsular antigen (serologic test) ○ positive in CSF or blood specimens in >90% of patients with meningitis; generally specific ○ false-positive results may occur (usually if rheumatoid factor is also present) partner with RF testing TREATMENT For localized pulmonary disease: fluconazole For meningitis or other severe infection: amphotericin B (with or without flucytosine, followed by fluconazole) Organism Caps ule Germ tube Blastoconidia Arthroconidia Chlamydospore C. albicans - + + - + C. tropicalis - - + - V C. parasilosis - - + - - C. glabrata - - + - - C. neoformans + - + - - Geotrichum - - - + - T. beigelii - - + + - ASPERGILLOSIS Infection, usually of the lungs, caused by Aspergillus spp. Immunocompetent individuals may be asymptomatic Acquired through inhalation Manifestations Invasive lung infection (common) Pulmonary or sinus fungus ball ○ usually develop on the open spaces in the body ○ fungus ball / aspergilloma ⎼ an intact mycelium of aspergillus usually grows in the cavities in the lungs remnants of the damaged cells on the previous infections can also grow in the sinus area, ear canals ○ gradually enlarges and causes destruction of lung tissues especially around it Allergic bronchopulmonary aspergillosis → for individuals with asthma or cystic fibrosis, they develop allergic reaction with coughing, wheezing, and fever if the Aspergillus colonizes the lining of their airway Cerebral aspergillosis Disseminated types: (immunocompromised) ○ Keratitis, otomycosis ○ Onychomycosis ○ Sinusitis, endocarditis, CNS infection @mlstranses | 4 MACROSCOPIC rapid grower (2-6 days) fluffy to granular, white to blue green colonies [LEFT] Aspergilloma on MRI [RIGHT] Cerebral aspergillosis ZYGOMYCOSIS or MUCORMYCOSIS An infection caused by a diverse group of fungal organisms either from the order Mucorales Rhizopus, Rhizomucor, Mucor, Absidia, Cunninghamella Symptoms may frequently result from invasive necrotic lesions in the nose and palate, pain, fever, orbital cellulitis, purulent nasal discharge CNS symptoms may follow Etiologic Agents - Aspergillus spp. Aspergillus fumigatus (representative organism) ○ most common agent isolated in immunocompromised individuals Aspergillus flavus Aspergillus niger ○ commonly isolated from fungus ball and otitis externa Aspergillus terreus ○ difficult to isolate Pulmonary symptoms are usually severe and includes productive coughing, high fever, and dyspnea Decaying vegetable matter, old bread or soil Inhalation Rhinocerebral infection (nasal mucosa, palate, sinuses, brain) Lungs, GIT Less common than Aspergillus Individuals with DM or under immunosuppressive drugs has greater risk of acquiring the infection Pathogenesis Binds to fibrinogen and laminin in the alveolar basement membrane Produces gliotoxin (may inhibit phagocytosis) Neutrophils can adhere and kill the hyphae → but the fungal organism is a rapid grower; hence, they can overwhelm the neutrophils May invade pulmonary and vascular tissue ⇒ thrombosis and necrosis ⇒ hematogenous spread Laboratory Diagnosis: (A. fumigatus) MICROSCOPIC Septate hyphae that usually show dichotomous branching ○ branches usually emerged at 45 deg C from the parent hyphae Dome shaped vesicle with bottle shaped phialides Cutaneous form of Mucormycosis Rhinocerebral infection usually manifestation of Mucormycosis [Upper LEFT] H&E [ Upper RIGHT] Methenamine SIlver Etiologic Agents Generally called as: Zygomycetes ○ Rhizopus (R. oryzae) ○ Mucor ○ Absidia ○ Cunninghamella @mlstranses | 5 Laboratory Diagnosis: (Zygomycetes) MICROSCOPIC tissue specimens or exudates “lollipop-like” fruiting body large ribbon-like branching, non septate hyphae and the presence of zygospore sac-like sporangia connected to the sporangiophore Rhizoids ⎼ hyphal structure that act like roots Stolons ⎼ hyphal structure that act like connectors ○ part of the hyphae will grow away from the main branch, then land on another area to grow another set of fruiting bodies CUNNINGHAMELLA With stolons Pyriform sporangia Funnel shaped vesicle (APOPHYSIS) Erect, straight, branching sporangiophores Globose or pyriform shaped vesicles One-celled, globose to ovoid sporangia C. bertholletiae MACROSCOPIC fluffy, white to gray to brown colonies 24-95 hours (rapid growers) grayish hyphae with brown to black sporangia ○ what is seen on expired bread ORGANISM MUCOR COLONY RHIZOPUS Sporangiophores (stem) Sporangia (bag of spores) Sporangiospores (inside the sporangia) No rhizoids and stolons Unbranched sporangiophores (B) Sporangia (A) Rhizoids appear at the point at which the stolon arises [UPPER L to R] Mucor and Absidia [LOWER L to R] Rhizopus and Cunninghamella ABSIDIA Branched sporangiophores Sporangiophores arise between nodes from which rhizoids are formed @mlstranses | 6  HYALOHYPHOMYCOSIS Fusarium ○ common environmental flora ○ several manifestations Mycotic keratitis (common) − occurs after traumatic corneal implantation ○ other forms: sinusitis, wound (burn) infections, allergic fungal sinusitis, RT secretions Laboratory Diagnosis MICROSCOPIC small septate hyphae large sickle-or boat-shaped macroconidia microconidia are produced (seldom) MACROSCOPIC grow rapidly (2-5 days) fluffy to cottony and may appear pink, purple, yellow, or green ○ F. oxysporum: purple ○ F. solani: pink TALAROMYCES Miscellaneous organism previously called Penicillium molluscum contagiosum-like ○ a cutaneous infection ○ molluscum contagiosum is a viral infection cutaneous ulcers of extremities other forms are bronchopulmonary, endocarditis T. marneffei – clinically significant species Laboratory Diagnosis MICROSCOPIC hyaline & septate hyphae brush-like conidiophores with metulae from which phialides with chains of conidia arise similar to Aspergillus but with minimal conidiation “skeleton fingers” MACROSCOPIC green, blue-green, white, pink colonies (saprophytic) typical yellow-green to pink colony with distinctive red diffusible pigment PNEUMOCYSTOSIS A lung infection Almost exclusively seen in individuals whose immune systems has been compromised by HIV or undergoing chemotherapy ○ for HIV/AIDS patient, pneumocystis is usually a terminal event Etiologic Agent Pneumocystis jiroveci (P. carinii) opportunistic atypical fungus causing pneumonia in immunocompromised hosts specimen: BAL fluid or lung biopsy does not grow artificially CHOLESTEROL in cell membrane Has cystic and trophozoite forms it falls between Ascomycetes and Basidiomycetes was previously identified as a protozoa but due to molecular studies, they are identified as true fungal organism @mlstranses | 7  Laboratory Diagnosis Cannot be cultured, can only be viewed on smears Trophozoite forms ○ pleomorphic ○ evident on GIEMSA Cystic forms ○ 4 to 7 um, does not bud, with intracystic bodies ○ methenamine silver; calcofluor white ; IF stain [LEFT] Methenamine Silver [RIGHT] Immunofluorescence stain Treatment Similar to TB patients Trimethoprim-sulfamethoxazole @mlstranses | 8