How to diagnose fungal infection

1. How to Diagnose
Fungus Diseases
Glenn S. Bulmer, Ph.D.
Prof. (hon.) Peking Medical University, Beijing

2. Types of Mycoses
• Superficial Mycoses
• Dermatophytoses
• Systemic Mycoses

3. Diagnosing Fungus Diseases
1. Clinical Clues
2. Culture of Etiologic Agent
3. Appearance in Tissue

4. 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

5. 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.

6. 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

7. 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

8. 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:

9. 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

10. Pulmonary cryptococcosis
PAS stain showing encapsulated
yeast in tissue

11. 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.

12. 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.

13. Small (3-5 microns)
intracellular yeast of
H. capsulatum
Blood smear showing
three intracellular yeast of
H. capsulatum

14. 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.

15. 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.

16. Note the numerous
intracellular yeast.
The infectious form of
Penicillium marneffei as
seen in nature or 24 C lab.
Dimorphic Penicillium marneffei

17. 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.

18. Dimorphic cultures of Sporothrix schenckii
Infectious form cultured at 24 C.
Pathogenic (yeast) cultured at 35 C

19. 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.

20. 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.

21. Dimorphic forms of Coccidioides immitis
PAS stain showing
sporangia in lung tissue.
Highly infectious spores
growing in soil or in the
laboratory.

22. 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.

23. 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.

24. Skin dermatophytosis: tinea corporis

25. Examples of tinea capitis and tinea pedis.

26. Tinea pedis and onychomycosis

27. KOH positive for hyphae. This confirms a
dermatophytosis but culture is necessary to identify fungus

28. Trichophyton rubrum. Most
common cause of ringworm
in China.
Microscopic of T.
mentagrophytes. Note large
(macroconidium) and small
spores (microconidia).

29. 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.

30. Two cases of pulmonary Aspergillosis
Infarct Aspergilloma

31. Aspergillosis or Phycomycosis?
Aspergillosis: Note
dichotomously branch,
septate hyphae.
Phycomycosis: Larger,
coenocytic hyphae.

32. c.) Phaeohyphomycosis
- Increasingly important systemic disease in China.
- Often seen forming abscesses.
- In tissue one sees dematiaceous, septate hyphae.

33. Phaeohyphomycosis
Young girl from Beijing with deep abscess. Not cured after
2 years of therapy. On the right is culture of etiologic agent.

34. 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.

35. Keratomycosis
Patient on left. Right is KOH of tissue containing numerous
hyaline hyphae.

36. 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.

37. Mycetoma cases
Note draining sinus tracts
from which granules are
obtained. Treated with
itraconazole.

38. Histopathology of Mycetomas
Actinomycotic mycetoma
granule. Note small (0.5
microns) filaments.
Eumycotic mycetoma granule.
Note pink coloured (PAS stain)
hyphae, 5 microns diameter.

39. 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.

40. Cases of Chromomycosis
(top) This case developed over 30
years and was seen before the advent
of itraconazole.
10-year old case

41. Fission bodies in Chromomycosis
Note the brown structures. These are
histopathology slides but they can be
seen readily in KOH preparation of skin.

42. 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.

43. 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.

44. Clinical aspects of Candidiasis
Trush Fatal candidiasis seen in
child lacking T-cells.

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

46. Clinical aspects of Candidiasis (con’t)
Massive gut erosion in
leukemic patient.
Placental candidiasis.

47. 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.

48. 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.

49. 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.

50. 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.

51. 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.

52. Thank You!