mycology systemic

8/8/2019 Mycology Systemic

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SYSTEMIC MYCOSESIntroduction

DIMORPHIC FUNGI

y Blastomycosis

o Blastomyces dermatitidis

y Coccidioidomycosis

o Coccidioides immitis

y Histoplasmosis o Histoplasma capsulatum

MONOMORPHIC FUNGI:

y Paracoccidioidomycosis

o Paracoccidioides brasiliensis

y Cryptococcosis

o Cryptococcus neoformans

Various appearances:

y Yeast cells:

o Histoplasma capsulatum

o Cryptococcus neoformans

y Sporangia


y Spherules


y Granules

o Sporothrix schenkii

Lab Dx:

o KOH and Culture

o Biopsy (Histopath)

o Immunoflourescence

o Immunohistochemistry

o Molecular study: PCR

Different stains used to demonstrate fungi in Histopath

1. PAS: bright red-purple

2. GMS: black

3. H & E: pink to purple

4. Mucicarmine: pink (Cryptococcus

5. Fontana-Masson: dematiaceous fungi

6. Calcoflour-white: fluorescence

7. Gridley: purple to magenta

BLASTOMYCES DERMATITIDIS

Distribution

endemic in

± North America

± Mississippi

± Missouri

± Ohio Rivers

± and their tributaries

zone extends into

± Kentucky

± Carolinas

± Appalachian region

± Canada

± and Wisconsin

Reservoirs

The exact ecologic niche has not been determined

but the fungus has been found in the following:

± moist environments

± wood

± tree bark

± rotting vegetation

± animal habitats

± manure

± wet acid soil from the banks of rivers

MOT

airborne routes

but is only rarely infectious

small threat to people with competent immune

systems

although it can cause fatal infections to immuno-

compromised patients

Human Infection

Blastomycosis

± Gilchrist's disease / blastomycosis

± honoring the first man to publish reports of

the infection

± North American Blastomycosis

± a chronic granulomatous and suppurative

disease that may affect the following:

Skin and mucous membrane

Bones

Lungs

GUT

Cutaneous Blastomycosis

± ( skin) Blastomycosis

- elevated, macerated, ill-defined, scaly

borders, central ulcer

± ( mucous membrane and skin)

Blastomycosis

± Chronic cutaneous blastomycosis (with or

without lung involvement)

verrucose

ulcerated

suppurative

Systemic blastomycosis:

± involve any organ of the body or a

combination of organs

± Pulmonary Blastomycosis



Inhalation --- lungs ---

disseminated

Round pneumonia

"Mass" like density with

air bronchogram

± Osteoarticular Blastomycosis

Affects the spine, ribs, long bones

painful debilitating arthritis or

osteomyelitis

± GUT Blastomycosis

Affects the prostate and

epidydimis in males

Specimen Sources

skin scrapings

crusts from skin lesions

aspirated pus

Sputum

and other pulmonary specimens

oropharyngeal scrapings

biopsied tissue

blood

prostate secretions

Cerebrospinal Fluid

urine

Specimen Collection and Handling

Specimens should be collected aseptically according

to the standard protocol for the type of specimen

Transported to the laboratory without delay and

plated promptly

Direct examination:

10% KOH added to the mounting fluid to

clear the specimen before it is examined

Mucus and other thick substances should be treated

with N-acetyl-L-cysteine before the wet mount is

done

Tissue specimens:

fixed and stained with

H & E

GMS stain

Giemsa stain

Immunology

two antigens: A and B

A is reported to be the more useful of the

two

Currently no skin test for blastomycosis is

commercially available because of the lack of

purified antigens

Methods

Immunodiffusion (ID) tests - specificity of 84% to

100%, sensitivity of 57% to 62%

Enzyme Immunoassay (EIA) - sensitivity of 80%,

specificity of 98%

Complement Fixation (CF) method

Chemiluminescent DNA probe method

Special Precautions

Laboratory personnel who are working with any

fluffy white colony should take special precautions

and adhere stringently to laboratory procedures.

As a minimum, cultures should always be studied

within a biologic Class II safety cabinet.

Slide cultures should never be made

Culture Media

Modified SDA or BHIA with antibiotics at

temperatures from 25° to 30°C,

SDA without antimicrobials at temperatures above

30°C

Use BHIA containing blood for culture of specimens

from body sites that are normally sterile

Biphasic culture bottles containing both BHI broth

and BHI agar are recommen-ded for blood cultures

PDA or PFA may be used for subculture to encourage

conidia production

Temperature Considerations

Mould form:

Optimal temperature for growth is 25° to

30°C on routine media

Yeast form:

37°C is needed to induce the yeast phase

Primary cultures f or B. dermatitidis should be held

f or 4 to 8 w eeks be f ore being discarded as " no

grow th."

Macroscopic (Colony) Morphology

Mould-form colonies at 25°C

on modified SDA grow slowly

mature colonies typically grow in 6 to 21 days

white or beige to brown at first

with a waxy or glabrous texture

some isolates may be fluffy

are prickly in the center

Reverse pigment of the colonies is tan to brown



Later, colonies tend to become fluffy or woolly, and

some develop concentric rings.

At 37°C, colonies of yeast appear after 10 to 15 days

on enriched media such as BHIA with blood. They are white to light tan, with a wrinkled or

folded topography and a waxy texture.

Microscopic Morphology

Mold form

The thick, smooth walls of the conidia are

doubly re f ractile (double image)

In bad preparations Blastomyces

dermatitidis is difficult to identify because it

does not have unique structures. The round

conidia, septate, hyaline hyphae, and

sticklike conidiophores make it resemble a

number of other fungi. Conidia:

hyaline, pyriform or globose

average diameter of 6 um.

Borne directly or laterally

(lollipop)

The thick, smooth walls of the

conidia are doubly re f ractile

(double image)

Yeast form

At 37 oC

Hyaline

large (up to 20 um)

spherical to pyriform with thick walls

broad based budding yeast

"figure eight," or hourglass, configuration of

the parent-daughter combination

reproduce by forming buds that are

typically attached to the mother cell by

broad (4 to 5 um) necks.

Rarely the daughter cell buds without

separating from the parent, creating a

"threesome."

Stains

In GMS stain the wall of the organisms stains black.

In PAS stain the internal structure of the organism is

red.

Laboratory Identification

is a thermally dimorphic organism.

It can be converted from the mould form to the

yeast form (using enriched media at 37°C) to confirm

identification

Blastomyces can be identified more safely by the

specific

exoantigen test

DNA probe

E xo- Antigen or immunoidenti f ication

Simple

cost effective method of identifying or

confirming the suspected identity of the

organism, even in the absence of typical

morphology

Helpful Features for Identification of Blastomyces

dermatitidis:

Culture:

Mold form: white or beige to brown, waxy

or glabrous texture to fluffy colonies

Yeast form: waxy, wrinkled, light brown

colonies

Morphology:



o RT: lollipop-like mycelial forms

o 37C: thick-walled broad based budding

yeast (figure of 8or hour glass)

Organisms from Which Blastomyces dermatitidis Must Be

Differentiated

Cryptococcus neo f ormans

± Non-encapsulated

Paracoccidioides brasiliensis

± Detached buds

COCCIDIOIDES IMMITIS

Epidemiology

y first recognized in 1892 & as a fungal infection in

1900

y endemic in:

o Southwestern United States

o Mexico

o Central and South America

y incidence in the United States of 100,000

infections/yr.

y Reservoir:

o Alkaline desert soil

o Dust storms

o Desert rodents (vectors)

y Outbreaks are associated

o dust storms

o archeological digs

o climatic conditions o earthquakes

Unique Risk Factors

y Competent immune system:

o Relatively small threat

y More severe in:

o Dark skinned people

o Pregnancy

High levels of ESTRADIOL and

PROGESTERONE in pregnant

women account for an increased

risk of infection during pregnancy

y Occupational hazard:

o Construction workers

o Farmers

Human Infection

y Causes: Coccidioidomycosis

o aka: San Joaquin fever; Desert fever

y C. immitis is probably the most virulent of all agents

of human mycoses

o Very contagious

o requires biosafety level 3 cabinet

Forms of Coccidioidomycosis:

o Cutaneous Coccidioidomycosis

o Primary Pulmonary Coccidioidomycosis

Segmental pneumonia with LAD &

pleural effusion

CXR: egg-shaped deformity

o Chronic Pulmonary Coccidioidomycosis

Hemoptysis due to cavitations

fatal

o Disseminated Coccidioidomycosis

Fatal

Meningitis & hydrocephalus:

y most common

complication

y Common cause of

mortality

o Coccidioidomycosis in AIDS patients

- acquired by AIDS patients

exposed to endemic areas

- pulmonary in form

Life cycle of Coccidioides

Specimen source

y sputum

y pulmonary specimens

y aspirated pus

y skin scrapings

y biopsied tissue

y CSF

y blood

y urine

Laboratory Diagnosis:

Immunology

y 2 substances used as antigen



o Coccidioidin: Is a filtrate prepared from

mould cultures

o Spherulin: Extract from a tissue culture of

the yeast form

y Skin test

o conversion from a negative to a positive

skin test is diagnostic of infection

y CF

o quite specific, but a few cross-reactions

with other mycotic infections

y TP Test

o Highly specific with very few cross-reactions

y LA

o being reevaluated because of reports of up

to 10% false-positive results with CSF and

diluted sera.

y DNA probes

o for culture confirmation

y exoantigen test

o to speed identification of the fungus using

indirect FA

y FA Tests

o rare cross-reaction

o excellent screening test

Culture Media

y Modified SDA

y SDA with antimicrobials

y BHIA

y BHIB

y Temperature

o Optimum temp. = 25-35oC for the mould

form.

o Tissue culture = 37-40oC with increased

CO2.

o Tmax = 54C

Macroscopic Morphology

y colonies are white and floccose at first

y Mature colonies o white to gray, but strains with lavender,

buff , cinnamon, yellow or brown pigment

o Reverse: tan to dark brown or orange

o Texture: Powdery

y membranous or glabrous with hyphae that are

adherent to the agar

y Surface = Partially or completely covered with a

cottony aerial mycelium that resembles COBWEB

Mold colony

y


y Hyphae o septate and hyaline

o RACQUET hyphae may also be observed in

culture

y Disjunctors (disjuncture cells)

o contains the arthroconidia

o they fragment when mature, freeing the

arthroconidia to disperse

y Arthroconidia

o single celled, barrel-shaped or rectangular

o May round up in tissues --- spherules

o Mature: contains endospores or asteroid

bodies

Direct Examination

y In host tissues

o Spherules are round and refractive and

usually have thick walls.

o Endospores are freed when the mature

spherule ruptures

o Can be seen in intact or rupturing spherules

in tissue sections and other specimens

Coccidioides immitis

barrel-shaped arthroconidia



Hyphae and developing spherules ( in lung tissue)

Coccidioides immitis:

arthroconidium; ruptured spherule

Helpful features for identification:

y Rapidly growing colonies with early appearance of

white cottony aerial mycelium and areas of adherent

surface hyphae

y Barrel-shaped arthroconidia alternating with

disjuncture cells at 25 to 35oC

y Spherules containing endospore in tissue or at 40C in

special media

CRYPTOCOCCUS NEOFORMANS

y Aka: Torulosis, European Blastomycosis

Introduction:

y 22 strains; 5 serotypes (A-D & AD);

y 3 human variants:

o neoformans (AIDS &

immunocompromised)

o gatti, grubi (non-immunocompromised)

y WORLDWIDE distribution pigeon roosts

y Viable for 2 years or more loses capsule

y MOT: inhalation of yeast cells

y Pathogenesis: inhaled alveolar spaces of hosts

lung, establish colonies and produce a capsule --- BV

--- CNS

y Sources:

o Var. neoformans: weathered pigeon

droppings

o Var. gatti: Eucalyptus tree (red gum)

o Contaminated dairy products, fruits and

vegetables,

Unique Risk Factor

y Opportunistic pathogen, exposure to soil

contaminated with pigeons excrement.

Forms of Cryptococcosis:

y Cutaneous Cryptococcosis

o painless pustules/papules/nodules,

hemorrhagic, waxy, umbilicated & ulcerated

y Pulmonary Cryptococcosis

o Acute: immunocompromised patients

asymptomatic to mild flu-

like S/Sx

o Chronic: lobar pneumonia, cavitations due

to production of granulomas with

encapsulated fungi at the center

y Disseminated Cryptococcosis

o mainly CNS: Subacute/Chronic meningitis

o S/Sx: papilledema, visual loss, seizures,

hydrocephalus

o Other organs: endopthalmitis,

chorioretinitis, conjunctivitis, sinusitis,

pericarditis, gastritis, bone infection

Cutaneous cryptococcis

(f ace: papules)

Cutaneous cryptococcosis ( arm: hemorrhagic/ulcerated

nodules)

Cutaneous cryptococcosis ( extremities)



Cutaneous cryptococcosis (f ace: plaque)

Subcutaneous cryptococcosis ( arm)

C T w ith intravenous contrast medium injection. Nodular cystic

lesion w ith enhancing capsule and central core, in the parietal

region on the le f t side.

Laboratory Diagnosis

y Specimen Sources:

Tissue, CSF, blood, urine,

respiratory secretions, pus

and or biopsied tissue

from skin lesions

y Direct Examination:

Concentrate specimens by

filtration or centrifugation

Filtration preferred

(centrifugation may

collapse the yeast cells,

making them inviable;

aerosol formation)

India ink preparation (not

sensitive but fast)

Immunology

y Four Serotypes:

o Serotype A most human infections

o Serotype B US West Coast, AIDS - rare

o Serotype C tropical areas* (Philippines)

o Serotype D Europe

y Measuring Antibody Titers:

o Fluorescent Antibody Test

tissue studies & serotyping

cultures

o Whole Yeast Cell Tube Agglutination Test

and EIA

Cryptococcus in serum

*E arly diagnosis o f in f ection - improves prognosis

( appropriate treatment can be started)

y Antigen Test: (more specific)

o Rapid LA Test (simple)

polysaccharide capsular antigen

in serum and CSF

- cross reaction with rheumatoid

factor or disseminated

Trichosporon beigelii

o infections (remedy: treat serum with

Na2EDTA or 5 mg of pronase/mL of serum)

o significant titer: >1:2 active

cryptococcosis

Culture Media and Temperature Considerations

y Media:

o Modified SDA

o Niger Seed Agar

o Dopamine agar

o (w/ or w/o antibiotics sensitive to

cycloheximide)

Capsule production

enhanced on CAP @ 35oC,

5%-10% CO2 or 1%

peptone


y Modified SDA; 25oC-37

oC, 2-4d

o Dome shaped, shiny white to tan, yellow to

light pink or light brown

o mucoid colonies (+ capsule)



o Yeast form ONLY!!!

o *Dry and dull age


y Yeast:

o Thin-walled globose or oval-shaped

o Singly or in pairs with narrow points of

attachment between the mother and

daughter cells

o NO pseudohyphae nor true hyphae (very

rare strains rudimentary hyphae on CMT

agar and in tissue)

o refractile mucopolysacchardide capsule

Capsule is related to the

strength of the hosts

immune response

Capsules form to protect

the organism from the

host

o India ink preparation

fungus in CSF, replaced by LA test

for capsular antigen (greater speed

and sensitivity)

India ink prep good technique,

viewing encapsulated yeasts in

culture

Diagramatic Structure

Cryptococcus: India Ink

Cryptococcus ( E M )

Cryptococcus: L A stain

Cryptococcus: H & E

Cryptococcus: Mucicarmine stain

Laboratory Identification

y special methods for examining microscopic

morphology

o Biochemical tests:



Carbohydrate and nitrate

utilization:

dextrose, maltose, sucrose,

galactose cellobiose, inositol,

xylose, raffinose, trehalose,

dulcitol, starch

Urease production +

Phenoloxidase test +

brown colonies in Caffeic acid/bird

seed/thistle/niger seed agar

Treatment

o Non-immunocompromised:

Amphotericin B + Flucytosine

o Immonucompromised:

Amphotericin B + Flucytosine +

Fluconazole

o AIDS px:

longer tx; Fluconazole as

maintenance

PARACOCCIDIOIDES BRASILIENSIS

y A.K.A. Paracoccidioidomycosis or South American

Blastomycosis

y P. brasiliensis, hyaline hyphomycete

y Endemic: Holdridge Life Zones, northwestern,

central, and southeastern South America, Central

America, and southern Mexico

y Reservoir & Unique Risk Factors

o Saprobic mould form acid soil in humidareas (endemic)

o Plants

o Armadillos - carrier

y MOT: Airborne (i.e., plants)

y Risk factors:

o Adult males combined effect of hormonal

makeup and occupations

o Malnutrition and immunocompetence

Human Infection/s

y Pathogenesis:

o Primarily an oral lesion (mouth, palate,

nasal) --- BV & lymphatics --- disseminated

esp to the lungs

y Primary Pulmonary Paracoccidioidomycosis

asymptomatic or subclinical

o self -limiting

subacute primary pulmonary disease mild changes

in the lungs; positive skin test

Secondary asymptomatic infections - pulmonary

disease or dissemination

y Disseminated disease in any organ (esp GIT)

y Pyogenic abscesses and ulcers - granulomatous

y Lymphadenitis - common

Specimen Sources

y Sputum (other pulmonary specimens)

y Pus aspirated from lymph nodes

y Skin scrapings or biopsied tissue edge of ulcers

y Biopsied lung tissue

y Crusts from skin lesions

Specimen Collection and Handling

y Aseptic technique

y Direct examination:

o Simple wet mounts, with added stains

o KOH clear debris

o N-acetyl-L-cysteine (NALC) mucus and

thick substances

o Tissue specimens (fixed and stained)

Papanicolaou stain, H&E, or Giemsa stain

o Specific FA Test detect yeast form in

tissue

Immunology

y Paracoccidioidin - E2 antigen extract from yeast

1. Precipitin Test band 1

2. Skin Tests intradermal injection

epidemiological tool

first serologic test to be positive

do not differentiate between past

exposure and current condition

negative skin test person who was

previously positive indicates the

anergy of disseminated infection

3. CF Test

a. yeast filtrate antigens: recommended

serologic test (titers appear in late and

remain detectable for several months

curedb. cross reactions low titers in patients with

acute histoplasmosis and blastomycosis.

4. ID Test

o concentrated yeast antigens and reference

sera are available

o Sensitivity, 94% and highly specific

o (+) one to three precipitin continuous bands

or identical with the reference sera is

indicative of infection



o Band 1 is the first to disappear during

treatment

o number of bands is correlated with the CF

titer

o Low titers: localized infection

o high titers: acute infection or dissemination.

o When symptoms are present a combination

of ID and CF tests is 98% specific for

diagnosis of paracoccidioidomycosis.

5. Exoantigen Methods

a. useful for speeding the identification of

cultures

6. Direct FA Tests

a. detects P. brasiliensis cells in smears of

clinical materials

Culture Media and Temperature Considerations

y Modified SDA with antimicrobials except

cycloheximide (primary isolation contaminated with

bacteria; slows fungus growth) at 25oC to 30oC

y SABHI and BHIA with blood (sterile sites)

y No antibiotics above 30C

y Yeast Extract Agar used for primary culture to

encourage initial growth and conidiation

y PDA and PFA for subculture to encourage

conidiation


y Mould Form; 25oC, Mod. SDA

o Slow maturation 2 cm in diameter after 2

to 3 weeks

y Young: White to cream, with short, downy aerial

mycelia and elevated centers.

y Mature: Flat, with a membranous or velvety texture

and cerebriform or folded topography.

y Pigment: Beige or brown, with a yellow-brown

reverse in mature colonies.

y Mycelial growth at 24°C on Mycosel Agar, 6 weeks of

incubation. Note multiple colonies.

Microscopic MorphologyMould Form; 25

oC to 30

oC

y Modified SDA

y Hyphae: Very fine, hyaline and septate

y Conidia: Few small oval to pyriform truncate on

short conidiophores or sessile hyphae

y Chlamydoconidia: Terminal and intercalary, w/

racquet and coiled hyphae

Yeast Form:

y Yeast Extract Agar (deficient in glucose)

y Conidia: Large number of oval to pyriform with

thick-walled arthroconidia in alternating pattern

y Yeast Form; 37C

o Large spherical to pyriform cells with thick

walls

o Reproduce by multiple budding with buds

that cover the entire surface of the parent

cell... MARINERs WHEEL appearance

o BUDS: Attached by thin necks and easily

dislodged.

o mariners w heel

Microscopic: septated hyphae and chlamydospore f ormation

( cotton-blue preparation) (40 X )

Y east culture at 36° C in trypticase soy agar. Observe multiple

budding yeast cells characteristic o f Paracoccidioides

brasiliensis ( cotton blue preparation) (40 X ).

Helpful Features

y Slow-growing colonies white cottony aerial

mycelium and heaped-up topography with

membranous or velvety @ 25oC,

y Folded colony of yeast cells @ 37oC,

y Large, thick-walled, multiple budding yeast cells

(mariners wheel) at 37oC



H ISTOPLASMA CAPSULATUM

Epidemiology

y Phil: 1992 1st reported case

y Prevalent in:

o Central North America

o Central South America

o Africa

o Australia

o India

o Malaysia

y Darlings disease

Reservoir

y Soil with high nitrogen content

y Caves

y birds droppings (birds, chicken, bats)

Human Infection

y Histoplasmosis y MOT: inhalation

y CXR:

o Fibrosis

o coin lesions

Forms of Histoplasmosis

1. Acute Pulmonary Histoplasmosis

y 1-3 wks incubation

y Non-specific flu-like S/Sx; (-) AFS;

y resolves w/o tx

y CXR: infiltrates to pleural effusion

y complications:

o aseptic arthritis/arthralgia

o erythema multiforme

o LAD

2. Chronic Pulmonary Histoplasmosis

y no LAD, no pleural effusion

y assoc with COPD

y Patho: pneumonia --- fibrosis --- cavitation --- lung

destruction --- pleural thickening

y CM: cough, hemoptysis, pleuritic pain

y CXR: interstitial infiltrates in apex of lungs

3. Disseminated Histoplasmosis

y seen in AIDS and immunocompromised patients

y involves the tongue, lungs, liver, GIT, adrenal glands,

blood

y Treatment: Amphotericin B, Itraconazole

Lab Diagnosis

1. Microscopic examination

2. Culture

3. immunology

Specimen

y pulmonary specimen

y pus / abscess

y skin scrapings

y biopsied tissues

y bone marrow

y blood

y csf and urine

Direct Examination

y intracellular

o Wright-Giemsa

o not KOH

o histology lab

H & E

Paps stain

y Mucous (NALC)

y FA tests

Culture Media

y BAP

y mod SDA: moist

y BHIA / BHIB: from sterile sites

y Yeast Extract PO4: inhibits candida

y PDA: encourages conidiation

y Primary cultures should be held for 10 to 12 weeks

before discarding as no growth


y very fine hyphae

y septate and hyaline

y ropelike in BA

y microconidia on short conidiophore

y macroconidia

o on short hyphae

o conidiophore at right angle

o pyriform

o echinulate

At 25 to 30 degree celsius



y sunflower in bloom

y resembles Candida and Blastomyces

y blastoconidia are small and globose

y smaller end of mother cell

y narrow neck of attachment

At 37 degree celsius

Immunology

y histoplasmin (H and M Ag)

y skin test

y CF test

o 1:8 and 1:32

o histoplasmin/yeast extract

o 2 3 weeks

o 4 fold change

y ID and CIE tests

o precipitin bands

o H active

o M early/after recovery

y RIA

o in reference labs

o highly sensitive

o low specificity

o detects histoplasma polysaccharide antigen

y Exoantigen test

o detects H and M Ags

o faster method

o identical bands

test and reference

Helpful features

slow intermediate growth at 250C

sunflower tuberculate macroconidia at RT

waxy, wrinkled, tan yeast at 370C

small blastoconidia with narrow neck at 370C

intracellular yeast in RES tissue, BM and WBC

histopath: granuloma, giant cells (diff dx: TB)

Conversion of mycelial form to yeast form

contributes to its virulence

D/dx

y Blastomyces dermatitidis

o nonbudding yeast

y Cryptococcus neoformans

o thinner capsule

y Paracoccidioides brasiliensis

o detached buds

y Coccidioides immitis

o endospores/young spherules

y Candida (Torulopsis glabrata)

o (-) Germ tube

mycology systemic

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