This document provides information on various laboratory techniques for the diagnosis of fungal infections. It discusses direct microscopic examination of clinical specimens, fungal culture techniques, serological and histological diagnosis methods, and newer non-cultural diagnostic methods like antigen detection, molecular diagnosis using DNA probes, and the use of Woods light. The key techniques covered are potassium hydroxide preparation, calcofluor white staining, fungal culture media and incubation, serological tests for fungal antibodies and antigens, histological staining methods, detection of fungal cell wall components like glucans and galactomannans, and molecular methods using DNA probes.

1. Name: Purshotam Kumar Sah Kanu
Roll No.: MB 1318/075
Level: M.Sc Microbiology (3rd Sem)
Central Department of Microbiology
Tribhuvan University, Kirtipur
Kathmandu, Nepal
MB 609 Systemic and Diagnostic
Mycology

2. Techniques of Diagnostic Mycology 5 hrs
Direct microscopic examination and culture, Serological
diagnosis, Histological diagnosis, Principles and applications
of molecular methods in the diagnosis of fungal infections

3. INTRODUCTION
 There are approximately 3,00,000 different species of
Fungi, of which only a few affect humans.
 These include aspergillosis, candidosis, coccidioidomycosis,
cryptococcosis, histoplasmosis, mycetomas, and
paracoccidioidomycosis.
 The successful laboratory diagnosis of fungal infection
depends majorly on the collection of appropriate clinical
specimens for investigations and also on the selection of
appropriate microbiological test procedures.

4. Laboratory Diagnosis
Laboratory diagnosis of fungal culcute depends on:
a) Direct microscopic examination
b) Culture
c) Serological diagnosis
d) Histological diagnosis
e) Non- cultural methods
f) Molecular methods

5. A) Direct Microscopy
 Direct microscopic examination depends on
demonstration of characteristic asexual spores,
hyphae, or yeast in various clinical specimens by light
microscopy.
 The commonly used clinical specimens are sputum,
lung biopsy material, and skin scrapings.
 The specimen is either treated with 10% KOH or
stained with special fungal stains.
 Use of 10% KOH dissolves tissue material, leaving the
alkali-resistant fungi intact.

6. CONT…
 Calcofluor dye is a fluorescent dye that combines with
fungal cell wall and is useful in identification of fungi
in tissue specimens.
 Methenamine silver stain is useful for demonstration
of fungi in tissues.
 India ink preparation of cerebrospinal fluid (CSF) is a
useful method for demonstration of white capsule of
C. neoformans in CSF.
 Gram staining is also useful to demonstrate Gram-
positive Candida species in the specimen.

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12. CONT….
 The disadvantages of microscopy are that it shows low
sensitivity and requires an experienced microscopist
for specific identification.

13. CONT….
A, This potassium hydroxide preparation of a skin scraping from a
patient with a dermatophyte infection shows septate hyphae
intertwined among epithelial cells. (Phase-contrast microscopy; 3500.)
B, This calcofluor white stain of urine
demonstrates Candida albicans.

14. CONT….
C, The deeply staining, small,
uniform yeast cells in this
histologic section of lung tissue
are typical of Histoplasma
capsulatum. (Methenamine silver
stain; 3430.)
D) India Ink Preparation

15. B) Culture
1. Culture media:
 Fungal culture is a frequently used method for
confirming the diagnosis of fungal infection.
 The commonest culture media used in mycology,
Sabouraud’s dextrose agar (SDA, pH 5.4), SDA with
antibiotics, potato dextrose or the slightly modified
potato flakes agar (PFA), and brain heart infusion
(BHI) agar with blood and antibiotics.
 Other media include CHROM agar, blood agar, etc.
 The low pH of the medium and addition of
chloramphenicol and gentamicin to inhibit bacterial
growth and cycloheximide (actidione) to inhibit
saprophytic fungi and thereby facilitate the
appearance of slow-growing fungi.

16. Cont…
• The pH of Emmons’ modification of SDA is close to
neutral and is more efficient medium for primary
isolation than the original formulation.
• 2. Incubation: Cultures are routinely incubated in
parallel at room temperature 25°C (room temperature
for weeks) and at 37°C for days.
• Many fungi develop relatively slowly and cultures
should be retained for at least 2-3 weeks (in some
cases up to 6 weeks) before being discarded. Yeasts
usually grow within 1-5 days.

17. Cont…
 Fungal colony is identified by rapidity of growth, color,
and morphology of the colony at the obverse and
pigmentation at the reverse.
 Microscopy of the fungal colony is carried out in
lactophenol cotton blue (LPCB) mount to study the
morphology of hyphae, spores, and other structures.
 The appearance of the mycelium and the nature of
the asexual spores are very much helpful to identify
the fungus.
 Culture, however, is time-consuming in most cases
and also the yield is not very good. Culture following
lysis of the specimens, such as blood, obviates this
problem.

18. Cont…
 Blood lysed by addition of certain substances,
followed by centrifugation, increases yield of fungi by
culture.
 Yield can be further increased with a shortening of
time by combining with BACTEC systems.

22. C) Serological diagnosis
 Demonstration of the antibodies in patient’s serum or
CSF is useful for diagnosis of fungal infections,
especially in systemic fungal infections.
 A significant rise of antibody titer in a paired sera
sample confirms the diagnosis.
 The complement fixation test was the earliest test
used in fungal serology and is still used in the
diagnosis of suspected cases of histoplasmosis,
blastomycosis, or coccidiomycosis.
 Recently, newer tests like ELISA (enzyme-linked
immunosorbent assay), Western blot, and
radioimmunoassays are increasingly used for
serodiagnosis of fungal infections.

23. CONT…..
•It has limited role.
•Used in diagnosis and follow up of Cryptococcus and
Candida with limits.
 The most common tests for:
a. Fungal antibodies:
1. Immunodiffusion;
2. Countercurrent immuno-electrophoresis (CIE);
3. Whole cell agglutination;
4. Complement fixation;
5. Enzyme-linked immunosorbent assay (ELISA).
b. Antigen detection:
1. Latex particle agglutination;
2. ELISA.

24. D) Histological diagnosis
 Common tissue stains used for detection of fungal
elements are the periodic acid-Schiff (PAS), Grocott-
Gomori methenamine-silver (GMS), hematoxylin and
eosin (H and E), Giemsa, and the Fontana-Masson
stains, are based on the presence of chitin and
polysaccharides in their cell wall.
 The Giemsa stain is used primarily to detect
Histoplasma capsulatum in blood or bone marrow
 PAS attaches to polysaccharides in the fungal wall and
stains fungi pink.
 The Fontana-Masson method stains melanin in the
cell wall and identifies the presence of dematiaceous
fungi.

25. Histopathology
Yeast cell: They
may be
intracellular small
yeast: e.g.
Histoplasma
capsulatum.
 They may have a
large distinguishing
capsule: e.g.
Cryptococcus.

26. Histopathology
•Spherules:
–Intact spherules are
large sac-like
structurefilled with
sporangiospores.
Left: A patient
showing the
disseminated stage
of disease
(coccidioidomycosi
s).
Top right:spherules.
Bottom right:chains
of arthrospores
interspersed with
empty cellular
compartments.

27. Histopathology
•Hyphae:
 –They may be brownin colour or non-coloured.
 –They may be septatedor non-septated.

28. Histopathology
•Granules: They
are tightly packed
masses of
hyphaeor
filaments,which
are surrounded by
tough outer rind.
Combination of
yeast cells and
hyphae:As in
Candida.

29. E)Noncultural methods
 These methods include (a) detection of fungal antigen,
(b) detection of fungal cell wall markers, and (c) detection
of fungal metabolites.
 Antigen detection: It is useful in immunocompromised
hosts where antibody detection is not as sensitive.
 Detection of fungal antigen in serum, CSF, and urine is
increasingly used for diagnosis of many fungal infections.
 Demonstration of antigen indicates recent or active
infection.
 Latex agglutination test is a frequently used test to
demonstrate polysaccharide capsular antigen of C.
neoformans in CSF for diagnosis of cryptococcal meningitis.
 False-positive reactions due to Trichosporon beigelli
and Capnocytophaga canimorsus are known.

30. Cont…
 Detection of fungal cell wall markers: Mannan is a highly
immunogenic component of the candidal cell wall.
 Mannan antigen detection, therefore, is most widely used
method in the diagnosis of candidiasis.
 Galactomannan is a heat-stable heteropolysaccharide
found in the cell walls of all Aspergillus species.
 Production of the galactomannan antigen is proportional
to fungal load in tissue, hence is being used as the
prognostic marker for diagnosis of invasive aspergillosis.
 A sandwich ELISA using rat monoclonal antibody EB-A2
against galactomannan antigen is being currently used in
Europe for diagnosis of invasive aspergillosis.

31. Cont….
 Most pathogenic fungi have 1, 3-beta-D-glucan in their cell
walls and minute quantities are secreted into the
circulation during the life cycle. Detection of this antigen
can also be used as an indicator of invasive fungal
infections.
 Detection of 1, 3-beta-D-glucan is based on its ability to
activate a coagulation cascade within amebocytes derived
from the hemolymph of horseshoe crabs.
 This uses a different cascade than endotoxin to cause
coagulation, hence is specific for fungi.
 The test does not detect certain species, such as C.
neoformans and Zygomycetes.

32. Cont….
 Detection of fungal metabolites: Detection of
distinctive fungal metabolites is another approach for
the diagnosis of fungal infections. Gas liquid
chromatography is being used to quantify arabinitol
for diagnosis of C. albicans infections.

33. F) Molecular Diagnosis
 DNA probes are the recent techniques, which are very
useful to identify colonies growing in culture at an
earlier stage of growth.
 These DNA probes are very useful for rapid diagnosis
of these cultures in comparison to traditional methods
of visual detection of colonies.
 DNA probes are now available for detection of
Cryptococcus, Histoplasma, Blastomyces, and
Coccidioides.
 Mitochondrial DNA has been used for the diagnosis of
C. albicans and Aspergillus species.

34. G) Woods light:
•Helps in clinical
diagnosis.
•Long wave ultraviolet
rays (black rays) which
when come in contact
with mycoticareas of
skin and hair produce
fluorescent colours.
•Disadvantage: it
occurs in some
mycoticinfections only.

35. Fungal skin tests:
•It has no value in diagnosis.
•It does not differentiate
between active and past
infection.
•Mainly used for
epidemiological study.
•It is observed by formation of
indurationand swellingdue to
reaction between injected
antigen and T cells.
•e.g. Histoplasmin, Candidin,
Tricophytintests.