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Microbiology Systemic Mycoses Dr. Loyola February 28, 2013 4-6 !
Microbiology
Systemic Mycoses
Dr. Loyola
February 28, 2013
4-6
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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:

 

Tissue

Nature

Coccydioidomycosis

spherules

hyphae

(Barrel shaped

Arthroconidia)

Histoplasmosis

Yeast (oval)

hyphae, microconidia and macroconidia (tuberculate chlamydospore)

Blastomycosis

Yeast (single

Hyphae (hyaline, branching septated) Conidia (slender terminal or lateral conidiophores)

bud, broad

base)

Paracoccidioidomyco

Yeast (mutliple

Hyphae and conidia (not distinctive)

sis

buds, narrow

base)

 

Dimorphism

Dimorphic fungi grow as yeasts or spherules (single-

celled forms that reproduce by budding) in vivo, as well as in vitro at 37°C, but as molds (multicellular hyphae) at 25°C.

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 25°C.

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

US, northern Mexico, Central and South America FEATURES Figure 1. Coccidioidomycosis life cycle Features: • In

Figure 1. Coccidioidomycosis life cycle Features:

In tissues (37°C):

o spherules filled with endospores

When cultured on Sabouraud’s agar at 25°C:

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)

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

young, cell-mediated immunity compromised - non-communicable Figure 2: Chronic cutaneous coccidioidomycosis showing

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

showing granulomatous lesions of the face, neck and chin Figure 3: Extension of pulmonary coccidioidomycosis •

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

(KOH; H&E) • spherules with endospores is diagnostic Figure 4: direct microscopy of skin scrapings from

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

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 25°C

- cultures with bacterial antibiotics and cycloheximide

arthroconidia with septated hyphae – highly infectious

spherule production in vitro by incubation in an enriched medium at 40°C, 20% CO 2

by incubation in an enriched medium at 40°C, 20% CO 2 Figure 6: Culture of Coccidioides

Figure 6: Culture of Coccidioides immitis

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)

Dimorphism:

37°C (tissue)

- yeast cell (hyphae and conidia convert to small, oval yeast cells)

25°C (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

lymph nodes, bone marrow) Primary cutaneous infection Figure 7: Histoplasmosis of the lower gum - ulcer

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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Figure 8: Tissue morphology of H. capsulatum - Histiocyte containing numerous yeast cells of Histoplasma

Figure 8: Tissue morphology of H. capsulatum

- Histiocyte containing numerous yeast cells of Histoplasma capsulatum

containing numerous yeast cells of Histoplasma capsulatum Figure 9: Tissue morphology of H. capsulatum - numerous

Figure 9: Tissue morphology of H. capsulatum

- numerous small narrow base budding yeast cells inside macrophages

2. Culture

mold at 25°C

conversion to yeast on an enriched medium at

37°C

• conversion to yeast on an enriched medium at 37°C Figure 10: small ovoid cells w/in

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

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

NOT REQUIRED for several cases

Acute pulmonary histoplasmosis

managed with supportive therapy and rest

Disseminated disease:

Amphotericin B

Itraconazole

Pulmonary lesions

- Surgical resection

BL

ASTOMYCOSIS

BL ASTOMYCOSIS

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 37°C

- Single bud is attached to the parent cell by a broad base

Mold at 25°C

- 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 blastomycin’s lack of sensitivity and specificity Pulmonary infection

Most common with symptoms indistinguishable from acute lower resp infection

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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

spleen • skin lesions on exposed areas are most common Figure 11: Typical Skin lession on

Figure 11: Typical Skin lession on Blastimycosis

skin – ulcerated verrucous granulomas with advancing borders and central scarring

granulomas with advancing borders and central scarring Figure 12: Ulcerated granuloma due to B. dermatitidis •

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)

yeast cells

bud is attached to the parent cell by a broad base

• bud is attached to the parent cell by a broad base Figure 13: Tissue section

Figure 13: Tissue section of Blastomycosis

2. Culture

Mold at 25°C

- Hyphae (hyaline, branching septated)

- Conidia (spherical, ovoid or piriform on a slender terminal or lateral conidiophores)

Conversion to yeast on an enriched medium at

37 o C

Colonies develop w/in 2 wks on SDA or enriched agar at 30˚C

In tissue or culture at 37˚C

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 37°C (in tissue)

o multiple budding yeasts; the buds are attached to the parent cell by a narrow base

at 25°C

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

37°C

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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

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

2.

Culture

 

mold at 25°C

conversion to yeast on an enriched medium at

 

37°C

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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