Microbiology

Systemic Mycoses
Dr. Loyola
February 28, 2013

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True Systemic (Endemic) Mycoses Coccidioidomycosis fungi exist in dry soil Histoplasmosis fungi exist in soil mixed with guano Blastomycosis Paracoccidioimycosis - Both agents presumed to reside in nature, but their habitats have not been clearly defined. GENERAL FEATURES Organisms thermally dimorphic fungi that exist in nature, dry soil geographic distribution varies (endemicity) Mode of Transmission inhalation pulmonary infection dissemination or direct cutaneous infection is deep No evidence of transmission among humans or animals caused by true pathogenic fungi capable of infecting healthy individuals, more severe in immunocompromised Each of these 4 mycosis is caused by a thermally dimorphic fungus and most infections are initiated in the lungs ff inhalation of the respective conidia With rare exceptions, these mycoses are not transmissible among humans or other animals Although most occur in immunocompetent individuals, the incidence among patients with aids and others with depressed cell mediated immunity is steadily increasing Dimorphic Systemic Mycoses These are fungal infections of the body caused by fungal pathogens which can overcome the physiological and cellular defenses of the normal human host by changing their morphological form. They are geographically restricted and the primary site of infection is usually pulmonary, following the inhalation of conidia. These are all saprophytic organisms that are acquired from the soil Animals do not serve as a direct zoonotic risk for human infection INFECTION 1. Spore Production Fungi are found in soil and in the feces of birds and bats The fungi produce spores that become airborne 2. Primary pulmonary infection When inhaled, spores cause a primary pulmonary infection 3. Dissemination of infection The fungi may travel from the lungs to other sites where infection can occur Possible sites of infection: Coccydioidomycosis o central nervous system, bone Histoplasmosis o Iiver, spleen, lymph nodes, bone marrow Blastomycosis o skin, bone, genitourinary tract Paracoccidioidomycosis o mucosa of the mouth and nose Dimorphism:
Coccydioidomycosis

Tissue spherules

Histoplasmosis

Yeast (oval)

Blastomycosis

Yeast (single bud, broad base)

Paracoccidioidomyco sis

Yeast (mutliple buds, narrow base)

Nature hyphae (Barrel shaped Arthroconidia) hyphae, microconidia and macroconidia (tuberculate chlamydospore) Hyphae (hyaline, branching septated) Conidia (slender terminal or lateral conidiophores) Hyphae and conidia (not distinctive)

Dimorphism Dimorphic fungi grow as yeasts or spherules (singlecelled forms that reproduce by budding) in vivo, as well as in vitro at 37C, but as molds (multicellular hyphae) at 25C. Dimorphism is regulated by factors such as temperature, CO2 concentration, pH, and the levels of cysteine or other sulfhydryl-containing compounds. Examples include Blastomyces dermatitidis (hyphae and yeast cells) and Coccidioides immitis (hyphae and spherules) The conversion of the mycelial form of Blastomyces dermatitidis to the large, globose, thick-walled, broadly based budding yeast form requires only increased temperature. Coccidioides immitis is a unique dimorphic fungus because it produces spherules containing endospores in tissue, and hyphae at 25C. o Increased temperature, nutrition, and increased carbon dioxide are important for the production of sporulating spherules. Histoplasma capsulatum o involves three stages o First stage, induced by an increase in temperature, respiration ceases and the level of cytochromes decreases. o Second stage of the mycelial-to-yeast conversion, cysteine or other sulfhydryl-containing compounds are required. o Final stage is characterized by normal cytochrome levels and respiration as the yeast grows and reproduces. Paracoccidioides brasiliensis o In tissue the yeast is characterized by multiple buddinG

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COCCIDIOIDOMYCOSIS Etiology: (Coccidiodes immitis) Location: confined to southwestern US, northern Mexico, Central and South America FEATURES

Secondary (Disseminated) Infection (1%) Risk Factors: Hereditary, sex, age, compromised CMI - Racial groups: Filipinos, African Americans, Native Americans, Hispanics, Asians chronic/fulminant infection of lungs, meninges, bones and skin - male more susceptible (with exeption of pregnant women- diff in immune response and sex hormones) has estrogen binding proteins, estradiol and progesterone stimulates its growth - greater risk in old and young, cell-mediated immunity compromised - non-communicable

Figure 1. Coccidioidomycosis life cycle Features: In tissues (37C): o spherules filled with endospores When cultured on Sabourauds agar at 25C: o grows as a mold in 2-3 weeks; hyphae o barrel-shaped arthroconidia A spherule will develop endospores within, then break apart, releasing the endospores. This is the tissue form seen in pus or histological sections: spherules and loose endospores. o They can also be seen in a KOH preparation of sputum. o It is pathognomonic for coccidioidomycosis Desert soil, pottery, archaeological middens, cotton, and rodent burrows all harbor C. immitis. C. immitis is a dimorphic fungus with 2 life cycles. o The organism follows the SAPROPHYTIC cycle in the soil and the PARASITIC cycle in man or animals. The saprophytic cycle starts in the soil with spores (arthroconidia) that develop into mycelium. The mycelium then matures and forms alternating spores within itself. The arthroconidia are then released, and germinate back into mycelia. The parasitic cycle involves the inhalation of the arthroconidia by animals which then form spherules filled with endospores. The ambient temperature and availability of oxygen appear to govern the pathway PATHOGENESIS Inhalation of the infective particle, arthoconidia and spherule formation in vivo engulfment within phagosomes by alveolar macrophage activation of macrophages phagosome-lysosome fusion killing from the lungs, dissemination can occur to any organ of the body where fungi can invade & destroy the tissue (bone, CNS) Immune complex formation deposition leading of local inflammatory reactions immunosuppresion resulting from the binding of complexes to cells bearing Fc receptors CLINICAL FINDINGS Primary infection asymptomatic in most fever, chest pain, cough, arthralgia, headache (valley fever, San Joaquin Valley fever, desert rheumatism) nodular lesions in lungs (erythema nodosum or erythema multiforme)

Figure 2: Chronic cutaneous coccidioidomycosis showing granulomatous lesions of the face, neck and chin

Figure 3: Extension of pulmonary coccidioidomycosis large superficial, ulcerated plaque

DIAGNOSIS Samples sputum blood spinal fluid urine exudates tissue biopsiies (cutaneous lesions) 1. Direct examination (KOH; H&E) spherules with endospores is diagnostic

Figure 4: direct microscopy of skin scrapings from a cutaneous lesion are mounted in 10% KOH and Parker ink solution

- Endosporulating spherules of Coccidioides immitis

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Dimorphism: 37C (tissue) - yeast cell (hyphae and conidia convert to small, oval yeast cells) 25C (nature) - hyphae, microconidia and macroconidia (tuberculate chlamydospore) - hyphae and conidia are seen In nature, grows as a mold in association with soil and avian habitats At temp below 37C primary isolates develop brown mold colonies Grow slowly;requires inc of 4-12 weeks Hyphae produce microconidia and macroconidia with peripheral projection of cell wall materials In tissue or in vitro on rich medium at 37C, the hyphae and conidia convert to small, oval yeast cells In tissue the yeast are typically seen within macrophages (bec H capsulatum is a facultative intracellular parasite) Ecological niche of H. capsulatum is in blackbird roosts, chicken houses and bat guano. PATHOGENESIS Inhalation of microconidia/primary cutaneous inoculation Conversion to budding yeast cells Phagocytosis by alveolar macrophages Restriction of growth or dissemination to RES by bloodstream Suppression of cell-mediated immunity In tissue, the yeast are typically seen within macrophages, as H. capsulatum is a facultative intracellular parasite Initial inflammatory reaction becomes granulomatous CLINICAL FINDINGS Pulmonary infection Asymptomatic (95%) Mild,moderate, severe, chronic cavitary Disseminated infection RES (liver, spleen, lymph nodes, bone marrow) Primary cutaneous infection

Figure 5: Tissue section showing typical endosporulating spherules of Coccidioides immitis

- young spherules have a clear centre with peripheral cytoplasm and a prominent thick walled - endospores are later formed within the spherule by repeated cytoplasmic cleavage - rupture of the spherule releases endospores into the surrounding tissue where they re-initiate the cycle of spherule development 2. Culture Sabarouds Dextrose Agar (SDA) - mold colonies at 25C - cultures with bacterial antibiotics and cycloheximide arthroconidia with septated hyphae highly infectious spherule production in vitro by incubation in an enriched medium at 40C, 20% CO2

suede-like to downy, greyish white colony with a tan to brown reverse 3. Serology IgM test ( 2-4 wk Latex agglutination Test) IgG test (Immunodiffusion or Complement fixation) - Important Low titers do not rule out the presence of infection 4. Skin test (coccidioidin and spherulin antigens) Negative test - anergy (poor prognosis) - past primary infection - IP Spheruline is more sensitive in detecting reactors. TREATMENT Symptomatic primary infections - self-limiting For severe/systemic disease: - Amphotericin B - Itraconazole - Fluconazole (particularly for meningitis) Surgical resection of pulmonary cavities HISTOPLASMOSIS Etiology: (Histoplasma capsulatum Natural reservior: soil, bat and avian habitats Location: may be prevalent all over the world, but the incidence varies widely (most endemic in Ohio, Mississippi, Kentucky)

Figure 6: Culture of Coccidioides immitis

Figure 7: Histoplasmosis of the lower gum

- ulcer around base of the teeth Infection looks like TB however, histoplasmosis usually appears as bilateral interstitial infiltrateS

DIAGNOSIS Samples sputum, tissue, bone marrow, CSF, blood 1. Direct examination: Giemsa/Wright intra and extracellular yeast cells a positive direct microscopy demonstrating characterisitic yeast-like cells from any specimen should be considered significant small ovoid cells w/in macrophages in histo sections

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Etiology: Histoplasma capsulatum var. duboisii Differentiation from classical histoplasmosis: - larger, thick-walled yeast cells - pronounced giant cell formation in infected tissue - lesser pulmonary involvement - greater frequency of skin and bone lesions TREATMENT
Figure 8: Tissue morphology of H. capsulatum

NOT REQUIRED for several cases Acute pulmonary histoplasmosis managed with supportive therapy and rest Disseminated disease: Amphotericin B Itraconazole Pulmonary lesions - Surgical resection BLASTOMYCOSIS Etiology: (Blastomyces dermatitidis Location: - North America, Africa, South America, Asia (Human and dogs) - Rarely isolated from nature Also known as : North american blastomycosis Dimorphism: Yeasts at 37C - Single bud is attached to the parent cell by a broad base Mold at 25C - Hyphae (hyaline, branching septated) - Conidia (spherical, ovoid or piriform on a slender terminal or lateral conidiophores) In culture grows as a mold producing hyaline branching septate hyphae and conidia At 37C or in the host, it converts to a large, singly budding yeast cell Blastomyces dermatitidis survives in soil that contains organic debris (rotting wood, animal droppings, plant material) and infects people collecting firewood, tearing down old buildings or engaged in other outdoor activities which disrupt the soil. PATHOGENESIS Inhalation of infectious particles Infiltration of macrophages and neutrophils and granuloma formation Oxidative killing mechanisms of neutrophils and fungicidal activity of macrophages Primary cutaneous inoculation ANTIGENIC STRUCTURE Blastomycin lacks specificity and sensitivity because of cross reactivity Antigen A (more specific to Blastomyces sp.) Enzyme immunoassay BAD a cell surface and secreted protein that generates protective CMI response Initiated in the lungs Dissemination to other organs may occur but preferentially to skin and boneS CLINICAL FINDINGS Asymptomatic Infection Frequency unknown due to inadequate skin or serologic test because of blastomycins lack of sensitivity and specificity Pulmonary infection Most common with symptoms indistinguishable from acute lower resp infection

- Histiocyte containing numerous yeast cells of Histoplasma capsulatum

Figure 9: Tissue morphology of H. capsulatum

- numerous small narrow base budding yeast cells inside macrophages 2. Culture mold at 25C conversion to yeast on an enriched medium at 37C

Figure 10: small ovoid cells w/in macrophages in histo sections

3. Serology complement fixation titers remain positive after some years enzyme immunoassay more sensitive CF test for Ab to histolasmin or the yeast cells becomes positive in 2-5wks after infection In ID test: precipitins to 2 specific Ag are detected. o Ab to H antigen signifies active histosplasmosis while Ab to M antigen may arise from repeated skin testing or past exposure RIA or EIA for circulating Ag o Nearly all patients with disseminated histoplasmosis have a positive test for antigen in the serum or urine; Ag drops following a successful treatment and recurs during relapsE 4. Skin test (Histoplasmin antigen) limited diagnostic value Histoplasmin skin test becomes positive soon after infection and remains positive for years May become negative in disseminated histoplasmosis Repeated skin testing stimulates serum Ab in sensitive individuals interfering with diagnostic interpretation of serologic tests NOTE: Following initial infection most persons appear to develop some degreee of immunity Immunosuppression may lead to reactivation and disseminated disease

African Histoplasmosis

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 Histologic examination : distict pyogranulomatous reaction with neutrophils and noncaseating granuloma Chronic Cutaneous Infection subcutaneous nodule, ulceration Disseminated Infections skin, bone, GUT, CNS, spleen skin lesions on exposed areas are most common

yeast cells bud is attached to the parent cell by a broad base

Figure 13: Tissue section of Blastomycosis

2. Culture Mold at 25C - Hyphae (hyaline, branching septated) - Conidia (spherical, ovoid or piriform on a slender terminal or lateral conidiophores) Conversion to yeast on an enriched medium at 37oC
Figure 11: Typical Skin lession on Blastimycosis

skin ulcerated verrucous granulomas with advancing borders and central scarring

Colonies develop w/in 2 wks on SDA or enriched agar at 30C In tissue or culture at 37C When grown on SDA at room temperature, whitish or brown colony develops with branching hyphae bearing conidia on slender terminal or lateral conidiophores; larger chlamydosphores may also be produceD 3. Serology Immunodiffusion test Complement fixation ELISA to detect antibodies to antigen A - ( = severity) High antibody titers - associated with progressive pulmonary or disseminated infection 4. Skin test (Blastomycin antigen) limited/no diagnostic value TREATMENT Amphotericin B Itraconazole Fluconazole Corrective surgery PARACOCCIDIOIDOMYCOSIS ETIOLOGY: (Paracoccidioides brasiliensis LOCATION: Central and South America ( rarely isolated in nature) ALSO KNOWN AS: South American blastomycosis DIMORPHISM: Produce chlamydospores and conidia at 37C (in tissue) o multiple budding yeasts; the buds are attached to the parent cell by a narrow base at 25C o hyphae and conidia o ship-stering wheel appearance - due to presence of multiple buds PATHOGENESIS inhalation of conidia more common in males (90%), 30-60 years old DETERMINANTS OF PATHOGENECITY the fungus has a protein in its cytoplasm which binds only to estrogen but not to testosterone - this binding prevents conversion to yeast form at 37C

Figure 12: Ulcerated granuloma due to B. dermatitidis

Cutaneous blastomycosis haematogenous spread gives rise to cutaneous lesions in over 70% of patients painless and present either as raised verrucous lesions with irregular borders, or as ulcers most frequent sites involved: face, upper limbs, neck and scalp Chronic blastomycosis the skin is the most common extrapulmonary site of infection manifest as ulcerated granulomas infection is acquired by the respiratory route primary site of infection is the lung Pumonary infiltrate is the most common presentation associated with other symptoms fever, malaise, cough, myalgia, night sweats Also present with chronic pneumonia When dissem occurs, skin lesions on exposed surfaces are common; may evolve into ulcerated verrucous granulomas with advancing border and central scarring Blastomycosis o Not as common in immunocompromised patients (including those with AIDS) o Possible sites of infection: skin, bone, genitourinary tract DIAGNOSIS Samples sputum tissue 1. Direct microscopic exam (KOH, H&E)

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 yeast cell wall polysaccharides (alpha-glucan) - stimulate granuloma formation Initial lesions occur in the lung CLINICAL FINDINGS initial lesions occur in the lung after a period of dormancy (duration variable), pulmonary granulomas may become active leading to chronic, progressive pulmonary disease or dissemination SYMPTOMATIC INFECTIONS nodular lesions in lungs dissemination to other organs many patients present with painful oral sores histology revelas either granulomas with central caseation or microabscesses MUCOCUTANEOUS PARACOCCIDIOIDOMYCOSIS a. ulcerated lesion on the pharyngeal mucosa b.extensive destruction of facial features A common triad of symptoms that are seen in Latin America is pulmonary lesions, edentulous mouth and cervical lymphadenopathy DIAGNOSIS Samples sputum tissue 1. Direct microscopic examination (KOH, H&E) multiple budding yeasts; the buds are attached to the parent cell by a narrow base

Figure 14: Ship-steering wheel appearance due to presence of multiple buds

2. Culture mold at 25C conversion to yeast on an enriched medium at 37C 3. Serology Immunodiffusion Complement fixation Healthy people in endemic area (-) titer = increased severity Serologic testing is most useful for diagnosis Ab to paracoccidiodin can be measured by CF or ID test In patients, titer correlate with severity of the diseas TREATMENT Itraconazole Appears to me most effective Amphotericin B Ketoconazole Sulfonamides Amphotericin b severe disease Itraconazole appears to be most effectivE

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