3.) how to diagnose fungus diseases

8/8/2019 3.) How to Diagnose Fungus Diseases

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How to DiagnoseHow to Diagnose

Fungus DiseasesFungus Diseases

Glenn S. Bulmer, Ph.D.

Prof. (hon.) Peking Medical University, Beijing


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Types of MycosesTypes of Mycoses

Superficial Mycoses

Dermatophytoses

Systemic Mycoses


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Diagnosing Fungus DiseasesDiagnosing Fungus Diseases

1. Clinical Clues

2. Culture of Etiologic Agent

3. Appearance in Tissue


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1.) Clinical Clues:

a. Chronic, slowly evolving

b. History: soil/airborne skin and lungs

c. Compromised: genetic or induced

d. Clinical picture only suggestivee. Serology & chemistry: of little value in

mycology


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2.) Culture of Etiologic Agent:

a. Sabourauds (SAB) is most useful medium.

Incubate at 25r C, rarely 35r C.

b. Sabourauds + antibiotics (Mycosel) for

dermatophytes and non-yeast pathogens.

c. Potato dextrose agar or blood agar are cheap and

useful.

d. Brain heart infusion agar used to culture yeast

phase at 35r C.


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3.) Appearance in Tissue

a. Direct Examination: KOH examination of tissue

(10 or 20%)

b. Histopathology:

Periodic Acid-Schiff is best;

Silver excellent (e.g., GMS);

H & E good for tissue but poor for fungi


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3.) Appearance in Tissue (cont.)

It has been my experience that fungi causing human

diseases are seen in tissue in one of six different ways:

1.) Yeasts

2.) Sporangia

3.) Hyphae

4.) Granules

5.) Fission (sclerotic) bodies

6.) Yeast and hyphae together


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Fungi in Tissue (cont.)

1.) Yeasts: These vary in size, shape, method of

dividing, with of without a capsule, etc.

The following illustrates several distinguishing

features of yeasts and the diseases they cause:


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a.) Only one pathogenic yeast has a capsule. The disease it

causes is called Cryptococcosis:

- Fatal disease of brain (CSF), causing meningitis

- encapsulated yeast seen in India ink

- In pigeon droppings and near Eucalyptus trees

- fluconazole and itraconazole

- 5 cases/million normal population but >20%AIDS


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Pulmonary cryptococcosis

PAS stain showing encapsulated

yeast in tissue


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C. neoformans culture grown

at either 24r C or 35r C.

Organism is monomorphic.

C. neoformans as seen in

culture or in CSF. Note huge

capsule.


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b.) Two mycoses have intracellular yeast. One of these is

Histoplasmosis and the other is Penicilliosis.

Histoplasmosis

- Granulomatous disease of lungs and RES whichmimics TB.

- Spread from bird droppings, especially blackbirds,

chickens and bats.

- Worldwide, 10% people China skin test positive (very

high in Sichuan).

- Hard to diagnose, use itraconazole.


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Small (3-5 microns)intracellular yeast of

H. capsulatum

Blood smear showingthree intracellular yeast of

H. capsulatum


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Infectious form of

Hi stoplasma capsulatumshowing spores. In nature

or lab at 24r C.

Yeast (pathogenic) form as in

vivo or cultured at 35r C.

This is a dimorphic fungus.


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The second intracellular yeast causes Penicilliosis. This is

a relatively new disease that is found exclusively in S.

China (south of Yangtze, from Guangdong to Yunnanprovinces) and S.E. Asia. It is the number 3 cause of

death for AIDS patients in Thailand.

Note characteristic target lesions ofpenicilliosis.


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Note the numerous

intracellular yeast.

The infectious form of

Penicillium marneffei as

seen in nature or 24r C lab.

Dimorphic Penicillium marneffei


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Sporotrichosis is caused by another dimorphic yeast called

gardeners disease, acquired from plants with a scratchfrom plant thorns.

Characteristic lymphadenopathy. Patient on right has

secondary bacterial infection.


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Dimorphic cultures ofSporothrix schenckii

Infectious form cultured at 24r C.

Pathogenic (yeast) cultured at 35r C


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2.) Sporangia. These are large (20-40 microns), round

elements which contains numerous spores. They causecoccidioidomycosis which is endemic to SW United

States and Mexico. It is a fatal lung disease found

mostly in Asians and dark-skinned people. So far,

cases seen in Asia are imported.

Skin lesions in coccidioidomycosis.


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The organism Coccidioides immitis is found in desert soils as

shown here. This looks similar to areas in Southern Xinjiang

province. The spores become airborne, enter the lungs and

change into endospores.


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Dimorphic forms ofCoccidioides immitis

PAS stain showing

sporangia in lung tissue.

Highly infectious spores

growing in soil or in the

laboratory.


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3.) Hyphae. These are the long slender tubes bywhich most fungi grow. We see hyphae growing in

human tissue for several diseases. They may be 5-6

microns in diameter or up to 10 microns in diameter

(depending upon the disease). Most are clear coloured

(hyaline) while others are brown (dematiaceous). Some

are septate while others are coenocytic (no septa).

The following are some diseases where we see hyphae

in tissue. Note some distinguishing features.


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a.) Dermatophytoses

- Often these diseases are referred to as: tinea + body

location; athletes foot; jock itch; or simply

ringworm.- These diseases maybe spread from man to man, animal

to man and soil to man.

- Most are characterized by the presence of clear

(hyaline), septate hyphae which is 5-6 microns indiameter.

- KOH (10-20%) preparations of skin hair or nails are

used for a preliminary diagnosis.


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Skin dermatophytosis: tinea corporis


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Examples of tinea capitis and tinea pedis.


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Tinea pedis and onychomycosis


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KOH positive for hyphae. This confirms a

dermatophytosis but culture is necessary to identify fungus


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Trichophyton rubrum. Most

common cause of ringwormin China.

Microscopic ofT.

mentagrophytes. Note large

(macroconidium) and small

spores (microconidia).


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b.) Aspergillosis and Phycomycosis (Zygomycosis,

Mucormycosis)

- Chronic or rapidly fatal: see hyaline, filamentous fungi

- Organisms in environment, cannot eliminate.

- Predisposed patients, worldwide

- Diagnosed by histopathology or repeated culture.

- No good serology tests.

- Therapy very difficult.


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Two cases of pulmonary Aspergillosis

Infarct Aspergilloma


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Aspergillosis or Phycomycosis?

Aspergillosis: Note

dichotomously branch,

septate hyphae.

Phycomycosis: Larger,

coenocytic hyphae.


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c.) Phaeohyphomycosis

- Increasingly important systemic disease in China.

- Often seen forming abscesses.

- In tissue one sees dematiaceous, septate hyphae.


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Phaeohyphomycosis

Young girl from Beijing with deep abscess. Not cured after

2 years of therapy. On the right is culture of etiologic agent.


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d.) Keratomycosis (mycotic keratitis)

- Many fungi in environment can cause

infection of outer portion of the eye. If not

treated patient will go blind or organism willdisseminate to the brain.

- Diagnosed by observing hyaline hyphae in

KOH eye scrapings.


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Keratomycosis

Patient on left. Right is KOH of tissue containing numerous

hyaline hyphae.


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4.) Granules. These are relatively large (1-2 mm) very

hard structures that are produced in draining sinus

tracts. They are only seen in cases of mycetoma.Mycetomas are caused by numerous genera of higher

bacteria (actinomycotic mycetoma) or true fungi

(eumycotic mycetoma). This is important because

depending upon the etiology they are treated

differently.


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Mycetoma cases

Note draining sinus tracts

from which granules are

obtained. Treated with

itraconazole.


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Histopathology ofMycetomas

Actinomycotic mycetoma

granule. Note small (0.5

microns) filaments.

Eumycotic mycetoma granule.Note pink coloured (PAS stain)

hyphae, 5 microns diameter.


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5.) Fission (sclerotic) bodies. These are round, brown

structures that are 15-20 microns in diameter. They are

not yeast cells or hyphae. They appear to divide bysplitting in the middle (fission).

The etiologic agent are all dematiaceous fungi which

live in the soil. The organism enters the body

following a puncture wound.


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Cases ofChromomycosis

(top) This case developed over 30

years and was seen before the advent

of itraconazole.

10-year old case


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Fission bodies in Chromomycosis

Note the brown structures. These are

histopathology slides but they can be

seen readily in KOH preparation of skin.


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6.) Yeast + Hyphae. In only one major mycosis do we

see a combination of yeast and hyphae in tissue. This

disease is candidiasis and it is the most importantmycosis in the world today.


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Important characteristic of Candidiasis:

- Endogenous in origin. Controlling predisposing

factors may be more important that specific therapy.

- The major mycosis of immunocompromised patients,

e.g., cancer, IVs, underlying diseases, surgery, acute

illnesses, age, excessive use of antimicrobials and

steroids, depress CMI, major trauma, diabetes, etc.

- 90% of AIDS patients have candidiasis.


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Clinical aspects ofCandidiasis

Trush Fatal candidiasis seen in

child lacking T-cells.


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Clinical aspects ofCandidiasis (cont)

Candidiasis of the neck

Onychomycosis caused by

a Candida sp.


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Massive gut erosion in

leukemic patient.

Placental candidiasis.


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Cancer patient whodied of candidiasis.

Numerous white focal

points are candidiasis.

Kidney from rabbit injectedwith steroids and Candida

albicans from the authors

mouth. Died in 4 days.


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Histopathology ofCandidiasis

The dark blue elements (B

& B stain) are hyphae and

yeast in candidiasis.

This is a PAS stain of

candidiasis. All the fungal

elements are pink.


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Identification ofCandida spp.

One week old culture of

C. albicans on

Sabourauds agar

Germ tube test: universallyused to identify C. albicans.

Inexpensive and requires

only 1-2 hours incubation in

serum.


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CHROMagar identification method

Candida spp. are identified on this medium by color change.

Also, can determine if patient has a mixed infection. Thismedium is available in China.

Also used in China is the API test which identifies species

biochemically.


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Conclusions

1.) Clinical aspects are of little value in

diagnosing mycoses except for the

dermatophytoses and sporotrichosis.

2.) KOH and histopathology of tissues is animportant diagnostic tool. Almost all

mycoses can be diagnosed and therapy

initiated immediately.

3.) Culture of disease agent is necessary to

prove etiology. This requires 2-4 weeks

incubation and a knowledgeable technician.


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Thank You!Thank You!

3.) how to diagnose fungus diseases

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