How to Diagnose

Fungus Diseases

Glenn S. Bulmer, Ph.D.

Prof. (hon.) Peking Medical University, Beijing
Types of Mycoses

• Superficial Mycoses
• Dermatophytoses
• Systemic Mycoses
Diagnosing Fungus Diseases

1. Clinical Clues
2. Culture of Etiologic Agent
3. Appearance in Tissue
1.) Clinical Clues:

a. Chronic, slowly evolving

b. History: soil/airborne – skin and lungs
c. Compromised: genetic or induced
d. Clinical picture – only suggestive
e. Serology & chemistry: of little value in
mycology
2.) Culture of Etiologic Agent:

a. Sabourauds (SAB) is most useful medium.

Incubate at 25 C, rarely 35 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 35 C.
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
3.) Appearance in Tissue (con’t.)

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
Fungi in Tissue (con’t.)

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:
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
 PAS stain showing encapsulated
yeast in tissue
Pulmonary cryptococcosis
 C. neoformans culture grown
at either 24 C or 35 C.
Organism is monomorphic.

C. neoformans as seen in
culture or in CSF. Note huge
capsule.
b.) Two mycoses have intracellular yeast. One of these is
Histoplasmosis and the other is Penicilliosis.

Histoplasmosis
- Granulomatous disease of lungs and RES which
mimics 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.
 Small (3-5 microns)
intracellular yeast of
H. capsulatum

Blood smear showing

three intracellular yeast of
H. capsulatum
Infectious form of
Histoplasma capsulatum
showing spores. In nature
or lab at 24 C.

Yeast (pathogenic) form as in

vivo or cultured at 35 C.
This is a dimorphic fungus.
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 Yunnan
provinces) and S.E. Asia. It is the number 3 cause of
death for AIDS patients in Thailand.

Note characteristic “target” lesions of

penicilliosis.
 Dimorphic Penicillium marneffei

Note the numerous

intracellular yeast.

The infectious form of

Penicillium marneffei as
seen in nature or 24 C lab.
Sporotrichosis is caused by another dimorphic yeast called
“gardener’s disease”, acquired from plants with a scratch
from plant thorns.

Characteristic lymphadenopathy. Patient on right has

secondary bacterial infection.
 Dimorphic cultures of Sporothrix schenckii

Infectious form cultured at 24 C.

Pathogenic (yeast) cultured at 35 C

Fungi in Tissue (con’t.)
2.) Sporangia. These are large (20-40 microns), round
elements which contains numerous spores. They cause
coccidioidomycosis 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.

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.
 Dimorphic forms of Coccidioides immitis

PAS stain showing

sporangia in lung tissue.

Highly infectious spores

growing in soil or in the
laboratory.
Fungi in Tissue (con’t.)

3.) Hyphae. These are the long slender tubes by

which 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.
a.) Dermatophytoses

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

location; athlete’s 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 in
diameter.
- KOH (10-20%) preparations of skin hair or nails are
used for a preliminary diagnosis.
Skin dermatophytosis: tinea corporis
Examples of tinea capitis and tinea pedis.
Tinea pedis and onychomycosis
 KOH positive for hyphae. This confirms a
dermatophytosis but culture is necessary to identify fungus
 Trichophyton rubrum. Most
common cause of ringworm
in China.

Microscopic of T.
mentagrophytes. Note large
(macroconidium) and small
spores (microconidia).
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.
Two cases of pulmonary Aspergillosis

Infarct Aspergilloma
 Aspergillosis or Phycomycosis?

Aspergillosis: Note
dichotomously branch,
septate hyphae.

Phycomycosis: Larger,
coenocytic hyphae.
c.) Phaeohyphomycosis

- Increasingly important systemic disease in China.

- Often seen forming abscesses.
- In tissue one sees dematiaceous, septate hyphae.
 Phaeohyphomycosis

Young girl from Beijing with deep abscess. Not cured after
2 years of therapy. On the right is culture of etiologic agent.
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 will
disseminate to the brain.
- Diagnosed by observing hyaline hyphae in
KOH eye scrapings.
 Keratomycosis

Patient on left. Right is KOH of tissue containing numerous

hyaline hyphae.
Fungi in Tissue (con’t.)
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.
 Mycetoma cases

Note draining sinus tracts

from which granules are
obtained. Treated with
itraconazole.
 Histopathology of Mycetomas

Actinomycotic mycetoma
granule. Note small (0.5
microns) filaments.

Eumycotic mycetoma granule.

Note pink coloured (PAS stain)
hyphae, 5 microns diameter.
Fungi in Tissue (con’t.)
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 by
splitting 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.
 Cases of Chromomycosis

(top) This case developed over 30

years and was seen before the advent
of itraconazole.

10-year old case

Fission bodies in Chromomycosis

Note the brown structures. These are

histopathology slides but they can be
seen readily in KOH preparation of skin.
Fungi in Tissue (con’t.)
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 important
mycosis in the world today.
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.
 Clinical aspects of Candidiasis

Trush Fatal candidiasis seen in

child lacking T-cells.
 Clinical aspects of Candidiasis (con’t)

Candidiasis of the neck

Onychomycosis caused by
a Candida sp.
 Clinical aspects of Candidiasis (con’t)

Massive gut erosion in

leukemic patient.

Placental candidiasis.
 Clinical aspects of Candidiasis
(con’t)
Cancer patient who
died of candidiasis.
Numerous white focal
points are candidiasis.

Kidney from rabbit injected

with steroids and Candida
albicans from the author’s
mouth. Died in 4 days.
 Histopathology of Candidiasis

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.
 Identification of Candida spp.

One week old culture of

C. albicans on
Sabourauds agar

Germ tube test: universally

used to identify C. albicans.
Inexpensive and requires
only 1-2 hours incubation in
serum.
 CHROMagar identification method

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

Also, can determine if patient has a mixed infection. This
medium is available in China.
Also used in China is the API test which identifies species
biochemically.
 Conclusions
1.) Clinical aspects are of little value in
diagnosing mycoses except for the
dermatophytoses and sporotrichosis.
2.) KOH and histopathology of tissues is an
important 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.
Thank You!